JP7015336B2 - Optical systems and assay chips for probing, detecting and analyzing molecules - Google Patents

Description

The present application relates to devices, methods, and techniques in general for performing rapid, massively parallel quantitative analysis of biological and / or chemical samples, and to methods of making said devices. ..

Detection and analysis of biological samples can be performed using biological assays (“bioassays”). Bioassays have traditionally required large and expensive laboratory equipment and research scientists trained to operate the equipment and perform bioassays. Moreover, bioassays have traditionally been performed in bulk, requiring large numbers of specific types of samples for detection and quantification.

Some bioassays are performed by tagging with a luminescent tag that emits light of a particular wavelength. The tag is illuminated by an excitation light source and causes luminescence, which is detected by the photodetector and quantifies the amount of luminescent light emitted by the tag.

Bioassays using luminescent tags have traditionally included expensive laser light sources for irradiating samples and complex, large detection optics and electronics for collecting luminescence from the irradiated samples. I need.

The techniques described herein relate to devices and methods for rapid analysis of samples using assay chips and instruments. The assay chip can be in the form of a disposable or recyclable chip, which is to accept a small amount of sample and to perform a large number of analyzes of the sample in the sample in parallel. It is configured in. Assay chips and instruments are used to detect the presence of specific chemical or biological analytical objects in some embodiments and chemical or biological reactions in some embodiments. And in some embodiments it is possible to determine the gene sequence. According to some embodiments, the integrated device can be used for single molecule gene sequencing.

According to some embodiments, the user deposits a sample in a chamber above the assay chip and inserts the assay chip into the receiving device. The instrument, alone or by communicating with a computer, automatically interfaces with the integrated device, sends and receives light from the assay chip, processes the received light, and provides the results of the analysis to the user. do.

According to some embodiments, the assay chip is a sample well configured to receive a sample, wherein the sample emits emission energy when excited. It includes at least one element that directs the emission energy in a particular direction and an optical path, which is the path along which the emission energy travels from the sample well toward the sensor. The at least one element is selected from the group consisting of a refraction element, a diffraction element, a plasmonic element, and a resonator.

In some embodiments, the assay chip is used in only a single biological assay prior to disposal.
In some embodiments, at least one element comprises at least one lens configured to direct emission energy towards the sensor.

In some embodiments, the at least one lens is a refracting lens.
In some embodiments, the optical path comprises at least one antireflection layer that is configured to reduce the reflection of emission energy at one or more interfaces of the assay chip.

In some embodiments, the assay chip further comprises a disposable frame.
In some embodiments, at least one element comprises concentric ring grating.

In some embodiments, concentric ring gratings are configured to increase the amount of excitation light from the excitation light source that couples into the sample well to excite the sample.

In some embodiments, concentric ring gratings are further configured to direct emission energy towards the sensor.
In some embodiments, the concentric ring grating is an aperiodic concentric ring grating.

In some embodiments, the at least one lens is a diffractive lens.
In some embodiments, at least one element consists of a dielectric resonator antenna.

According to some embodiments, the instrument is configured to interface with an assay chip containing multiple sample wells, where each sample well of the plurality of sample wells accepts a sample. The instrument is configured to excite at least a portion of a sample of a plurality of sample wells, with at least one source of excitation light and a plurality of sensors, each of the plurality of sensors. Sensors correspond to sample wells in multiple sample wells, and each sensor in multiple sensors is configured to detect emission energy from the samples in each sample well. With at least one optical element configured to direct the emission energy from each of the multiple sensors and each of the sample wells towards the respective sensor of the multiple sensors. including.

In some embodiments, the instrument reflects excitation light from at least one source of excitation light toward the assay chip, and emits energy from multiple samples towards multiple sensors. It further includes a polychromic mirror that is configured to be transparent.

In some embodiments, at least one optical element consists of a relay lens.
In some embodiments, the at least one excitation light source comprises a plurality of light sources, and each light source of the plurality of light sources is the excitation light at one or more of a plurality of wavelengths. Is released.

In some embodiments, the device further comprises a wavelength combiner for spatially superimposing the light emitted from each of the plurality of light sources.
In some embodiments, the at least one excitation light source comprises a pulsed light source.

In some embodiments, the instrument further comprises at least one spectral filter so that the at least one spectral filter allows emission energy to pass through, as well as excitation light from at least one excitation light source. Is configured to absorb and / or reflect.

In some embodiments, the instrument further comprises at least one spectral sorting element for spatially separating the first wavelength emission energy from the second wavelength emission energy.

In some embodiments, at least one spectral sorting element comprises a diffractive optical element.
In some embodiments, the diffractive optics both disperse the emission energies chromatically and focus the emission energies.

In some embodiments, the diffractive optics consist of an offset Fresnel lens.
In some embodiments, at least one spectral sorting element is an optical filtering element.

In some embodiments, the instrument further comprises a control system, which (i) directs the direct excitation light to the plurality of sample wells and (ii) the plurality of sensors. It is programmed to detect the signal from the well and (iii) use the spatial distribution pattern of the signal to identify the sample or its subunits.

According to some embodiments, the apparatus comprises an assay chip comprising a plurality of pixels and an instrument configured to interface to the assay chip. Each of the multiple pixels of the assay chip is a sample well configured to receive the sample, and the sample emits emission energy when excited, the sample well and the emission energy. At least one element for orienting in a particular direction, at least one element selected from the group consisting of a refraction element, a diffraction element, a plasmonic element, and a resonator, and an optical path. Emission energy travels along the optical path from the sample well towards the sensor, including the optical path. The instrument is a plurality of sensors, with at least one source of excitation light configured to excite the sample in each sample well, with each sensor of the plurality of sensors being a sample. Each sensor of multiple sensors corresponds to a well and is configured to detect the emission energy from the samples in each sample well, with multiple sensors and each sample well. Includes at least one optical element configured to direct the emission energy from the sensor towards each sensor of the plurality of sensors.

In some embodiments, the assay chip is configured to be connected to and removed from the instrument.
In some embodiments, the optical distance between the sample wells of the plurality of sample wells and the corresponding sensors of the plurality of sensors is less than 30 cm when the assay chip is connected to the instrument. be.

In some embodiments, the optical distance between the sample wells of the plurality of sample wells and the corresponding sensors of the plurality of sensors is less than 5 cm when the assay chip is connected to the instrument. be.

In some embodiments, the optical distance between the sample wells of the plurality of sample wells and the corresponding sensors of the plurality of sensors is less than 1 cm when the assay chip is connected to the instrument. be.

In some embodiments, the device is portable.
In some embodiments, each sample comprises a luminescent tag that emits emission energy within one wavelength band of the plurality of wavelength bands, and each sensor of the plurality of sensors has a plurality of wavelengths. Includes sub-sensors configured to detect emission energy in each of the bands.

In some embodiments, each sensor of the plurality of sensors comprises at least two sub-sensors.
In some embodiments, each sensor of the plurality of sensors comprises at least four sub-sensors.

In some embodiments, the instrument further comprises at least one wavelength dependent element, wherein the at least one wavelength dependent element is the first sub-sensor of the at least two sub-sensors with the emission energy of the first wavelength. Directs towards, and directs the emission energy of the second wavelength towards the second sub-sensor of at least two sub-sensors.

In some embodiments, the at least one wavelength dependent element is a diffractive optical element.
In some embodiments, the at least one wavelength dependent element is a spectral filter.

In some embodiments, at least one excitation source emits pulsed light.
In some embodiments, the first luminescent tag associated with the first sample is excited by the light of the first wavelength, but not by the light of the second wavelength, to the second sample. The associated second luminescent tag is excited by the light of the second wavelength, but not by the light of the first wavelength.

According to some embodiments, the method is a method of analyzing a sample.
Multiple sample wells, including providing the sample on the top surface of an assay chip consisting of multiple sample wells, and matching the chip to a device consisting of at least one excitation light source and at least one sensor. A step of exciting a sample from a sample in at least one of the wells with excitation light from at least one excitation light source and in response to excitation by the excitation light, in at least one sample well. It comprises the step of detecting the emission energy generated by the sample by at least one sensor.

In some embodiments, the method further comprises determining the type of molecule that has released the emission energy, based on the detection of the emission energy.
In some embodiments, the step of determining the type of molecule comprises measuring the properties of the emission energy spectrum.

In some embodiments, the step of determining the type of molecule further comprises determining the wavelength of the excitation light that excited the sample.
In some embodiments, at least one source of excitation light emits continuous wave light.

In some embodiments, at least one source of excitation light emits pulsed light.
In some embodiments, the at least one excitation light source comprises a plurality of excitation light sources, and each excitation light source of the plurality of excitation light sources emits light of a different wavelength.

In some embodiments, the sample comprises at least one nucleotide attached to the fluorophore.
According to some embodiments, the method for sequencing the target nucleic acid molecule is (a) providing the chip adjacent to the device including the excitation source and the sensor, the chip being at least one. The well comprises one well and at least one well is operably coupled to an excitation source and a sensor when the chip is in the sensing position of the instrument, and the well is a target nucleic acid molecule, a polymerizing enzyme, and. A step containing multiple types of nucleotides or nucleotide analogs, and (b) at the sensing position, the chip, in the presence of the polymerizable enzyme, at the priming site of the target nucleic acid molecule, undergoes an extension reaction to the nucleotide or nucleotide. In the step of sequentially incorporating the analog into a growing chain complementary to the target nucleic acid molecule, when integration and excitation by excitation energy from the excitation source occurs, the nucleotide or nucleotide analog is placed in the well. The step of emitting a signal and (c) the step of using a sensor to detect a spatial and / or temporal distribution pattern of a signal that is distinguishable with respect to multiple types of nucleotides or nucleotide analogs. , (D) The step of identifying a nucleotide or nucleotide analog based on the spatial and / or temporal distribution pattern of the signal, thereby sequencing the target nucleic acid molecule.

In some embodiments, a nucleotide or nucleotide analog comprises a tag, which emits a signal when incorporated into said growing strand.
In some embodiments, the tag is a luminescent tag.

In some embodiments, nucleotides or nucleotide analogs are identified following detection of spatial and / or temporal distribution patterns of said signals.
In some embodiments, the plurality of types of nucleotides or nucleotide analogs comprises four different types of nucleotides or nucleotide analogs and the spatial of said signal associated with four different types of nucleotides or nucleotide analogs. And / or temporal distribution patterns are distinguishable from each other.

In some embodiments, spatial and / or temporal distribution patterns of signals associated with the four different types of nucleotides or nucleotide analogs are detected separately from each other.

In some embodiments, the spatial and / or temporal distribution patterns of the signal are distinguishable from each other based on their respective shape and / or intensity distributions of the spatial and / or temporal distribution patterns. Is.

In some embodiments, the priming site comprises a primer complementary to the target nucleic acid molecule.
In some embodiments, the act (b) of the method of the invention comprises the step of carrying out a primer extension reaction using the primer, the primer being hybridized to the target nucleic acid molecule and said growth. Create a chain to grow.

In some embodiments, the target nucleic acid molecule is double-stranded.
In some embodiments, the priming location is a gap or nick in the target nucleic acid molecule.

In some embodiments, the polymerizing enzyme is immobilized in the wells.
In some embodiments, the polymerizing enzyme is immobilized at the bottom of the well.
In some embodiments, the polymerizing enzyme is immobilized using a linker attached to the surface of the well.

In some embodiments, the polymerizing enzyme exhibits chain substitution activity.
In some embodiments, the wells are between the plurality of wells in the chip.
In some embodiments, the instrument comprises a plurality of excitation sources operably coupled to said plurality of wells.

In some embodiments, a spatial and / or temporal distribution pattern of the signal is generated from the signal prior to the action (c) of the method.
According to some embodiments, the method for nucleic acid sequencing is (a) a step of providing a chip adjacent to a device, wherein the chip comprises a plurality of wells and the plurality of wells are chips. Are operably coupled to (i) the excitation source and (ii) the sensor of the device when is in the sensing position of the device, and the individual wells of the plurality are target nucleic acid molecules, polymerization. A step containing an enzyme and multiple types of nucleotides or nucleotide analogs, and (b) a growing chain complementary to the target nucleic acid molecule in the presence of the nucleotide or nucleotide analog and the polymerizable enzyme. In order to produce, in the step of subjecting the target nucleic acid molecule to a polymerization reaction by the chip at the sensing position, when excitation by excitation energy from the excitation source occurs during integration, the nucleotide or nucleotide-like. The body detects a step of emitting a signal into said individual well and (c) a spatial and / or temporal distribution pattern of the signal that is distinguishable for multiple types of nucleotides or nucleotide analogs. In order to do so, it comprises the steps of using a sensor and (d) identifying the sequence of the target nucleic acid molecule based on the spatial and / or temporal distribution pattern of the signal.

In some embodiments, the nucleotide or nucleotide analog comprises a tag, which emits the signal when incorporated into the growing chain.
In some embodiments, the tag is a luminescent tag.

In some embodiments, the sequence is identified following detecting the spatial and / or temporal distribution pattern of the signal.
In some embodiments, the spatial of said signal, wherein the plurality of types of nucleotides or nucleotide analogs comprises four different types of nucleotides or nucleotide analogs and is associated with four different types of nucleotides or nucleotide analogs. And / or temporal distribution patterns are distinguishable from each other.

In some embodiments, spatial and / or temporal distribution patterns of the signal associated with the four different types of nucleotides or nucleotide analogs are detected separately from each other.

In some embodiments, the act (b) of the method comprises the step of carrying out a primer extension reaction using a primer, which hybridizes to the target nucleic acid molecule to produce the growing strand. ..

In some embodiments, the target nucleic acid molecule is single-stranded.
In some embodiments, the polymerizing enzyme is immobilized in the well.
In some embodiments, the excitation source is operably coupled to the plurality of wells.

In some embodiments, the act (c) of the method comprises the step of detecting the spatial and / or temporal distribution pattern of the signal.
In some embodiments, the instrument comprises a plurality of excitation sources operably coupled to said plurality of wells.

In some embodiments, the device comprises a plurality of sensors operably coupled to said plurality of wells.
In some embodiments, a spatial and / or temporal distribution pattern of the signal is generated from the signal prior to the action (c) of the method.

The aforementioned and other embodiments, embodiments, and features of the present teaching can be more fully understood from the following description relating to the accompanying drawings.
The term "pixel" may be used in the present disclosure to describe a unit cell of an integrated device. The unit cell can include a sample well and a sensor. The unit cell can further include an excitation source. The unit cell is configured to enhance the coupling of excitation energy from the excitation source to the sample well, at least one excitation coupling optical structure, which is the "first structure". Can be referred to as). The unit cell can further include at least one emission coupling structure that is configured to enhance the coupling of emissions from the sample wells to the sensor. The unit cell can further include an integrated electronic device (eg, a CMOS device). It is possible that there are multiple pixels arranged in an array on top of the integrated device.

The term "optical" can be used herein to describe the visible, near-infrared, and short-wavelength infrared spectral bands.
The term "tag" as used herein refers to a tag, probe, marker, or reporter attached to a sample to be analyzed or attached to a reactant that can react with the sample. Can be used for.

The phrase "excitation energy" may be used in the present disclosure to describe any form (eg, radiant or non-radiative) of energy delivered to a sample and / or tag in a sample well. The radiation excitation energy can consist of optical radiation at one or more characteristic wavelengths.

The phrase "characteristic wavelength" may be used in the present disclosure to describe a central or dominant wavelength within a limited bandwidth of radiation. In some cases it is possible to represent the peak wavelength of the radiation bandwidth. Examples of characteristic wavelengths for fluorophores are 563 nm, 595 nm, 662 nm, and 687 nm.

The phrase "characteristic energy" may be used in the present disclosure to describe the energy associated with a characteristic wavelength.
The term "emissions" may be used in the present disclosure to describe emissions from tags and / or samples. It can include radiant emissions (eg, optical emissions) or non-radiant energy transfers (eg, Dexter energy transfer or Förster resonance energy transfer). Emissions result from excitation of the sample and / or tag in the sample well.

The phrase "emissions from sample wells" or "emissions from samples" may be used in the present disclosure to describe tags and / or emissions from samples within sample wells.

The term "self-aligned" is used herein in which a first graphic patterning step (eg, photolithography, ion-beam lithography, EUV lithography) prints a pattern of a first element and a second. At least without using two separate graphic patterning steps, where the graphic patterning step of 2 is aligned with the 1 lithographic patterning step and a prints the pattern of the 2nd element. Two separate elements (eg, sample well and emission coupling structure, sample well and excitation source) can be used to represent a microfabrication process that can be made and matched to each other. The self-aligned process can consist of including patterns of both the first element and the second element in a single lithography patterning process, or the first element is manufactured. It is possible to use the features of the structure to form a second element.

The term "sensor" as used herein refers to one or more integrated circuit devices configured to sense emissions from sample wells and produce at least one electrical signal representing the sensed emissions. Can be used to represent.

The term "nanoscale" is used herein to describe a structure having at least one dimension or minimum feature size on the order of 150 nanometers (nm) or less, but not greater than approximately 500 nm. obtain.

The term "microscale" can be used in the present disclosure to describe a structure having at least one dimension or minimum feature size between approximately 500 nm and approximately 100 microns.

The phrase "enhanced excitation energy" can be used herein to describe increasing the intensity of excitation energy in the excited region of a sample well. Intensity can be increased, for example, by concentrating and / or resonating the excitation energy incident on the sample well. In some cases, the intensity can be increased by an antireflection coating or lossy layer that allows the excitation energy to penetrate further into the excited region of the sample well. The excitation energy enhancement can be a comparative reference to an embodiment that does not include a structure that enhances the excitation energy in the excitation region of the sample well.

The terms "about," "approximately," and "substantially" can be used in the present disclosure to represent values and are intended to include reference values plus and minus acceptable variations. The amount of change can be less than 5% in some embodiments, less than 10% in some embodiments, and in some embodiments. It can be less than 20%. In embodiments where the device can function properly over a large range of values, for example a range containing one or more digits, the amount of change can be doubled. For example, "approximately 80" can include values between 40 and 160 if the device functions properly for values in the range 20-350.

The term "adjacent" can be used in the present disclosure to describe two elements that are placed very close to each other (eg, about 5 of the transverse or vertical dimensions of a pixel). Within a distance less than one-third). In some cases, intervening structures or layers can be present between adjacent elements. In some cases, adjacent elements can be directly adjacent to each other in the absence of intervening structures or elements.

The term "detect" is used herein to indicate that an emission from a sample well is received at a sensor and that it represents or produces at least one electrical signal associated with the emission. Can be done. Also, the term "detecting" is used herein to determine the presence of a particular sample or tag within a sample well, or within a sample well, based on emissions from the sample well. It can be used to represent identifying the nature of a particular sample or tag.

Those skilled in the art will appreciate that the figures described herein are for illustration purposes only. It should be understood that in some cases, various aspects of the invention may be exaggerated or expanded to facilitate understanding of the invention. In drawings, similar reference characters generally represent similar features, functionally similar elements, and / or structurally similar elements throughout the various figures. The drawings are not necessarily actual size and are instead emphasized in illustrating the principles of this teaching. The drawings are by no means intended to limit the scope of this teaching.

The figure which shows the emission wavelength spectrum by some embodiments. The figure which shows the absorption wavelength spectrum by some embodiments. The figure which shows the emission wavelength spectrum by some embodiments. Block diagram diagram of a device that can be used for rapid mobile analysis of biological and chemical samples, according to some embodiments. The figure which shows the schematic diagram of the relationship between the pixel of a sensor chip and the pixel of an assay chip by some embodiments. The figure which shows the component associated with a single pixel of an assay chip and a single pixel of a sensor chip by some embodiments. The figure which shows a part of the component of an apparatus by some embodiments. Top view of assay tip and tip holder frame according to some embodiments. Bottom view of assay chips and chip holder frames, according to some embodiments. The figure which shows the assay chip and the chip holder frame by some embodiments. The figure which shows the excitation energy incident on a sample well by some embodiments. The figure which illustrates the attenuation of the excitation energy along the sample well formed as a zero-mode waveguide according to some embodiments. In some embodiments, a diagram showing a sample well containing a dibot that increases the excitation energy in the excitation region associated with the sample well. FIG. 5 comparing excitation intensities for sample wells with and without divot according to one embodiment. The figure which shows the sample well and the divot formed in the protrusion by some embodiments. FIG. 5 shows sample wells with tapered sidewalls, according to some embodiments. FIG. 5 shows a sample well with curved sidewalls and dibots with smaller transverse dimensions, according to some embodiments. The figure which shows the sample well formed from the surface plasmonic structure. The figure which shows the sample well formed from the surface plasmonic structure. FIG. 6 shows a sample well containing an excitation energy-enhanced structure formed along the sidewall of the sample well, according to some embodiments. The figure which shows the sample well formed in the multi-layer stack by some embodiments. The figure which illustrates the surface coating formed on the surface of a sample well by some embodiments. The figure which shows the structure associated with the lift-off process which forms a sample well by some embodiments. The figure which shows the structure associated with the lift-off process which forms a sample well by some embodiments. The figure which shows the structure associated with the lift-off process which forms a sample well by some embodiments. The figure which shows the structure associated with the lift-off process which forms a sample well by some embodiments. The figure which shows the structure associated with the lift-off process which forms a sample well by some embodiments. The figure which shows the structure associated with the alternative lift-off process which forms a sample well by some embodiments. The figure which shows the structure associated with the direct etching process which forms a sample well by some embodiments. The figure which shows the structure associated with the direct etching process which forms a sample well by some embodiments. The figure which shows the structure associated with the direct etching process which forms a sample well by some embodiments. The figure which shows the structure associated with the direct etching process which forms a sample well by some embodiments. The figure which shows the sample well which can be formed in a plurality of layers using a lift-off process or a direct etching process by some embodiments. The figure which shows the structure associated with the etching process which can be used to form a divot by some embodiments. The figure which shows the structure which is associated with the alternative process which forms a divot by some embodiments. The figure which shows the structure which is associated with the alternative process which forms a divot by some embodiments. The figure which shows the structure which is associated with the alternative process which forms a divot by some embodiments. FIG. 6 shows structures associated with the process of depositing adherents and passivation layers, according to some embodiments. FIG. 6 shows structures associated with the process of depositing adherents and passivation layers, according to some embodiments. FIG. 6 shows structures associated with the process of depositing adherents and passivation layers, according to some embodiments. FIG. 6 shows structures associated with the process of depositing adherents and passivation layers, according to some embodiments. The figure which shows the structure associated with the process of depositing the adhering material in the center in a sample well by some embodiments. The figure which shows the surface plasmon structure only by one embodiment. The figure which shows the surface plasmon structure only by one embodiment. The figure which shows the surface plasmon structure formed adjacent to a sample well by some embodiments. The figure which shows the surface plasmon structure formed in the sample well by some embodiments. The figure which shows the surface plasmon structure formed in the sample well by some embodiments. The figure which shows the example of the periodic surface plasmon structure by some embodiments. The figure which shows the example of the periodic surface plasmon structure by some embodiments. The figure which shows the example of the periodic surface plasmon structure by some embodiments. The figure which shows the numerical simulation of the excitation energy in the sample well formed adjacent to the aperiodic surface plasmon structure by some embodiments. The figure which shows the periodic surface plasmon structure by some embodiments. The figure which shows the periodic surface plasmon structure by some embodiments. The figure which shows the periodic surface plasmon structure by some embodiments. The figure which shows the nano antenna which consists of the surface plasmon structure by some embodiments. The figure which shows the nano antenna which consists of the surface plasmon structure by some embodiments. The figure which shows the structure associated with the process process for forming a surface plasmon structure by some embodiments. The figure which shows the structure associated with the process process for forming a surface plasmon structure by some embodiments. The figure which shows the structure associated with the process process for forming a surface plasmon structure by some embodiments. The figure which shows the structure associated with the process process for forming a surface plasmon structure by some embodiments. The figure which shows the structure associated with the process process for forming a surface plasmon structure by some embodiments. The figure which shows the surface plasmon structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the surface plasmon structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the surface plasmon structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the surface plasmon structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the surface plasmon structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the surface plasmon structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the surface plasmon structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the surface plasmon structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the surface plasmon structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the surface plasmon structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the surface plasmon structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the surface plasmon structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the thin loss film formed adjacent to a sample well by some embodiments. The figure which shows the result from the numerical simulation of the excitation energy in the vicinity of a sample well and a thin loss film by some embodiments. The figure which shows the result from the numerical simulation of the excitation energy in the vicinity of a sample well and a thin loss film by some embodiments. FIG. 5 showing thin lossy membranes spaced apart from sample wells, according to some embodiments. FIG. 5 shows a stack of thin lossy membranes formed adjacent to a sample well, according to some embodiments. The figure which illustrates the reflection stack which can be used to form a resonance cavity adjacent to a sample well by some embodiments. The figure which shows the dielectric structure which can be used for concentrating the excitation energy in a sample well by some embodiments. The figure which shows the photonic band gap structure which can be patterned adjacent to a sample well by some embodiments. The figure which shows the photonic band gap structure which can be patterned adjacent to a sample well by some embodiments. The figure which shows the dielectric structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the dielectric structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the dielectric structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the dielectric structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the dielectric structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the dielectric structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the dielectric structure and the structure which is associated with the process process for forming a self-aligned sample well by some embodiments. The figure which shows the structure for coupling the excitation energy to a sample through a non-radiative process by some embodiments. The figure which shows the structure for coupling the excitation energy to a sample through a non-radiative process by some embodiments. The figure which shows the structure for coupling the excitation energy to a sample by a plurality of non-radiative processes according to some embodiments. The figure which shows the structure which incorporates one or more energy transformation particles in order to couple an excitation energy to a sample through a radiative process or a non-radiative process according to some embodiments. The figure which shows the spectrum associated with the down conversion of the excitation energy to a sample by some embodiments. The figure which shows the spectrum associated with the up-conversion of the excitation energy to a sample by some embodiments. The figure which shows the concentric plasmonic circular grating by some embodiments. The figure which shows the spiral plasmonic grating by some embodiments. The figure which shows the emission spatial distribution pattern from the concentric plasmonic circular grating by some embodiments. The figure which shows the plasmonic nano antenna by some embodiments. The figure which shows the plasmonic nano antenna by some embodiments. The figure which shows the plasmonic nano antenna by some embodiments. The figure which shows the plasmonic nano antenna by some embodiments. The figure which shows the refraction optics of an assay chip by some embodiments. The figure which shows the Fresnel lens of the assay chip by some embodiments. The figure which shows the microscope component of an instrument by some embodiments. The figure which shows the far-field spectrum sorting element of the sensor chip by some embodiments. The figure which shows the far-field filtering element of a sensor chip by some embodiments. The figure which shows the thin loss film of a sensor chip by some embodiments. The figure which shows the thin loss film of a sensor chip by some embodiments. The figure which shows the optical block of the device by some embodiments. The figure which shows the sensor in the pixel of the sensor chip in an elevation view by some embodiments. FIG. 5 shows a bullseye sensor with two separate and concentric active regions, according to some embodiments. The figure which shows the stripe sensor which has 4 separate active regions by some embodiments. FIG. 5 shows a quad sensor with four separate active regions, according to some embodiments. FIG. 5 shows an arc segment sensor with four separate active regions, according to some embodiments. The figure which shows the stacked segment sensor by some embodiments. The figure which shows the emission distribution from a sorting element with respect to the energy emitted at the 1st wavelength by some embodiments. The figure which shows the radiation pattern received by a bull's eye sensor corresponding to the emission distribution shown in FIG. 7-2A by some embodiments. The figure which shows the emission distribution from a sorting element with respect to the energy emitted at the 2nd wavelength by some embodiments. The figure which shows the radiation pattern received by a bull's eye sensor corresponding to the emission distribution shown in FIG. 7-2C by some embodiments. The figure which shows the result from the numerical simulation of the signal detection for the bullseye sensor which has two active regions with respect to the 1st emission wavelength from a sample by some embodiments. FIG. 6 shows the results from a numerical simulation of signal detection for the bullseye sensor associated with FIG. 7-2E with respect to a second emission wavelength from a sample, according to some embodiments. FIG. 6 shows the results from a numerical simulation of signal detection for the bullseye sensor associated with FIG. 7-2E with respect to a third emission wavelength from a sample, according to some embodiments. FIG. 6 shows the results from a numerical simulation of signal detection for a bullseye sensor associated with FIG. 7-2E with respect to a fourth emission wavelength from a sample, according to some embodiments. The figure which shows the result from the numerical simulation of the signal detection for the bullseye sensor which has 4 active regions with respect to the 1st emission wavelength from a sample by some embodiments. FIG. 6 shows the results from a numerical simulation of signal detection for the bullseye sensor associated with FIG. 7-2I with respect to a second emission wavelength from a sample, according to some embodiments. FIG. 6 illustrates a circuit above a device that can be used to read a signal from a sensor consisting of two active regions, according to some embodiments. FIG. 6 illustrates a three-transistor circuit that may be included in a sensor chip with respect to signal storage and readout, according to some embodiments. FIG. 6 illustrates a circuit above a device that can be used to read a signal from a sensor consisting of four active regions, according to some embodiments. The figure which shows the time emission property about two different emitters which can be used for a sample analysis by some embodiments. The figure which shows the temporal evolution of the light emission from an excitation source and a sample by some embodiments. The figure which shows the time delay sampling by some embodiments. The figure which shows the time emission characteristic about two different emitters by some embodiments. The figure which shows the voltage dynamics in the charge storage node of a sensor by some embodiments. The figure which shows the double lead of the sensor segment without a reset by some embodiments. FIG. 5 illustrates the first and second read signal levels associated with two emitters having temporally distinct emission characteristics, according to some embodiments. FIG. 5 illustrates the first and second read signal levels associated with two emitters having temporally distinct emission characteristics, according to some embodiments. The figure which shows the excitation spectrum band of the excitation source by some embodiments. The figure which shows the excitation spectrum band of the excitation source by some embodiments. FIG. 6 illustrates a method of operation of a compact device that can be used for rapid mobile analysis of biological and chemical samples, according to some embodiments. The figure which shows the calibration procedure by some embodiments. The figure which shows the data analysis procedure by some embodiments. The figure which shows the calculation environment by some embodiments.

The features and advantages of the present invention, when used in combination with the drawings, will become more apparent from the detailed description given below.
I. Recognizing the Challenges and Solutions by the Inventor The inventor recognizes that conventional equipment for performing bioassays is large and expensive and requires the implementation of advanced experimental techniques. got it. Many types of bioassays rely on the detection of a single molecule in a sample. Traditionally, single molecule detection may require a large, space-consuming laser system used to generate the high intensity light required for the excitation of molecules. In addition, a large optical component can be used to direct the laser beam to the sample, and an additional optical component can be used to direct the luminescent light from the sample to the sensor. Can be done. These conventional optical components may require precise alignment and stabilization. Conventional experimental equipment and the training required to use this conventional equipment can result in complex and expensive bioassays.

The inventor recognizes and recognizes that there is a need for devices that can easily and inexpensively analyze biological and / or chemical samples to determine the properties of their constituents. got it. Applications of such devices can be for sequencing biomolecules, such as nucleic acid molecules, or polypeptides with multiple amino acids (eg, proteins). A compact, high-speed device for performing single molecule or particle detection and quantification reduces the cost of performing complex and quantitative measurements of biological and / or chemical samples, and biochemistry. We were able to rapidly advance our technological discoveries in Japan. What's more, easily transportable, cost-effective devices have not only been able to change the way bioassays are performed in the developed world, but for the first time for people in developing regions, the ease of essential diagnostic testing. Access was able to be provided, which could dramatically improve health and welfare. For example, in some embodiments, devices for performing bioassays are used to perform diagnostic tests on biological samples such as blood, urine, and / or saliva. The device can be used by an individual in the home, by a doctor, or in a remote clinic in a developing country or any other place, such as a rural clinic. Such diagnostic tests can include the detection of biomolecules in a subject's biological sample, such as nucleic acid molecules or proteins. In some examples, diagnostic tests sequence nucleic acid molecules in a subject's biological sample, such as sequencing cell-free deoxyribonucleic acid molecules or expression products in the subject's biological sample. Including singing.

The term "nucleic acid" as a whole refers to a molecule consisting of one or more nucleic acid subunits, as used herein. The nucleic acid may include one or more subunits selected from adenosine (A), cytosine (C), guanine (G), thymine (T), and uracil (U), or variants thereof. It is possible. In some examples, the nucleic acid is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), or a derivative thereof. The nucleic acid can be single-stranded or double-stranded. The nucleic acid can be round.

The term "nucleotide" as a whole, as used herein, refers to a nucleic acid subunit, which may be an A, C, G, T, or U, or a variant thereof or a variant thereof. It is possible to include analogs. Nucleotides can include any subunit that can be integrated into the growing nucleic acid chain. Such subunits are A, C, G, T, or U, or are specific to or purin (ie, one or more complementary A, C, G, T, or U). Any other subunit complementary to A or G, or variants or analogs thereof) or pyrimidines (ie, C, T, or U, or variants or analogs thereof). It is possible. The subunit is degraded by an individual nucleobase or group of bases (eg, AA, TA, AT, GC, CG, CT, TC, GT, TG, AC, CA, or their uracil counterparts). Enables.

Nucleotides generally include nucleosides and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO ₃ ) groups. Nucleotides can include nucleobases, pentatosaccharides (either ribose or deoxyribose), and one or more phosphate groups. Ribonucleotides are nucleotides whose sugar is ribose. Deoxynucleotides are nucleotides whose sugar is deoxyribose. Nucleotides can be nucleoside monophosphate or nucleoside polyphosphoric acid. The nucleotide can be deoxyribonucleoside polyphosphate, such as, for example, deoxyribonucleoside triphosphate, which is deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine. It can be selected from triphosphate (dGTP), deoxyuridine triphosphate (dUTP), and deoxycytidine triphosphate (dTTP), dNTP, such as a luminescent tag or marker (eg, fluorophore), etc. Includes detectable tags.

Nucleoside polyphosphoric acids can have an "n" phosphate group, where "n" is a number of 2, 3, 4, 5, 6, 7, 8, 9, or 10 or greater. .. Examples of nucleoside polyphosphoric acids include nucleoside diphosphates and nucleoside triphosphates. Nucleotides can be terminal phosphate-labeled nucleosides, such as terminal phosphate-labeled nucleosides, polyphosphoric acids, and the like. Such a label may be a luminescent (eg, fluorescent or chemiluminescent) label, a fluorescent label, a colored label, a chromogenic label, a mass tag, an electrostatic label, or an electrochemical label. It is possible. The label (or marker) can be coupled to the terminal phosphate through a linker. The linker can include, for example, at least one or more hydroxyl groups, sulfhydryl groups, amino groups, or haloalkyl groups, which, for example, in the terminal phosphate of a natural or modified nucleotide, eg, phosphorus. It may be suitable for forming acid esters, thioesters, phosphoramidates, or alkylphosphonate linkages. The linker can be cleavable and is adapted to separate the label from the terminal phosphate, for example with the help of a polymerizable enzyme. Examples of nucleotides and linkers are provided in US Pat. No. 7,041,812, which is incorporated herein by reference in its entirety.

The term "polymerase" as a whole, as used herein, refers to any enzyme (or polymerizing enzyme) that can catalyze the polymerization reaction. Examples of polymerases include, without limitation, nucleic acid polymerases, transcription enzymes, or ligases. The polymerase can be a polymerizing enzyme.

The term "genome" generally refers to the entire genetic information of an organism. The genome can be encoded by either DNA or RNA. The genome can consist of coding regions that encode proteins and non-coding regions. The genome can contain the sequences of all chromosomes together in an organism. For example, the human genome has a total of 46 chromosomes. All these sequences together make up the human genome.

The present disclosure provides devices, systems, and methods for detecting biomolecules or subunits thereof, such as nucleic acid molecules. Such detection can include sequencing. The biomolecule can be extracted from a biological sample obtained from the subject. Biological samples can be extracted from the subject's body fluids or tissues, such as breath, saliva, urine or blood (eg, whole blood or plasma). Subjects may be suspected of having health conditions, such as illness (eg, cancer). In some examples, one or more nucleic acid molecules may be extracted from the subject's body fluids or tissues. One or more nucleic acids may be obtained from one or more cells obtained from a subject, such as part of a subject's tissue, or from a subject's cell-free body fluid, such as whole blood. It can be extracted from multiple cells.

Biological samples can be processed in preparation for detection (eg, sequencing). Such processing can include sequestration and / or purification of a biomolecule (eg, a nucleic acid molecule) from a biological sample, as well as the generation of more copies of the biomolecule. In some examples, one or more nucleic acid molecules are sequestered and purified from the subject's body fluids or tissues and amplified through nucleic acid amplification such as the polymerase chain reaction (PCR). One or more nucleic acid molecules or subunits thereof can then be identified, such as through sequencing.

Sequencing involves synthesizing another biomolecule that is complementary or similar to the template, eg, synthesizing a nucleic acid molecule that is complementary to the template nucleic acid molecule, and identifying nucleotide integration by time (ie,). , Synthetic sequencing) and the like can include determining individual subunits of a template biomolecule (eg, a nucleic acid molecule). As an alternative, sequencing can include direct identification of individual subunits of a biomolecule.

During sequencing, signals indicating individual subunits of the biomolecule can be collected in memory and processed in real time or at a later point in time to determine the sequence of the biomolecule. Such processing can include comparing the signal to the reference signal, which allows the identification of individual subunits, which in some cases produces leads. The read can be a sequence of sufficient length (eg, at least about 30 base pairs (bp)), which can be used to identify larger sequences or regions, eg, it is a chromosome. Or it can be aligned to a genomic region or location above the gene.

Sequence reads can be used to reconstruct longer regions of the subject's genome (alignment). Reads can be used to reconstruct a chromosomal region, an entire chromosome, or an entire genome. Sequence reads, or larger sequences originating from such reads, can be used to analyze the subject's genome, eg, to identify variants or polymorphisms. Examples of variants are, but are not limited to, single nucleotide polymorphisms (SNPs), including tandem SNPs, small nucleotide polymorphisms or insertions (also referred to as indels or deletion insertion polymorphisms or DIPs), and multiple nucleotide polymorphisms. Types (MNPs), columnar repeat sequences (STRs), deletions containing microdeletion, insertions containing microinsertions, structural mutations including duplication, inversions, translocations, proliferation, complex multisite mutation pairs, copies Includes number variation (CNV). Genome sequences can consist of combinations of varieties. For example, the genomic sequence can include one or more SNPs and one or more combinations of CNVs.

Individual subunits of a biomolecule can be identified using markers. In some examples, luminescent markers are used to identify individual subunits of a biomolecule. Some embodiments use a luminescent marker (also referred to herein as a "marker"), which can be an exogenous or endogenous marker. The extrinsic marker can be an external luminescent marker used as a reporter and / or tag for luminescent labeling. Examples of extrinsic markers are, but are not limited to, fluorescent molecules, fluorophores, fluorescent dyes, fluorescent dyes, organic dyes, fluorescent proteins, species, enzymes, and / or quantum involved in fluorescent resonance energy transfer (FRET). It is possible to include dots. Other extrinsic markers are also known in the art. Such extrinsic markers can be specifically conjugated to probes or functional groups (eg, molecules, ions, and / or ligands) that bind to a particular target or component. Attaching an extrinsic tag or reporter to the probe allows target identification through detection of the presence of the extrinsic tag or reporter. Examples of probes can include protein, nucleic acid (eg, DNA, RNA) molecules, lipids, and antibody probes. The combination of extrinsic markers and functional groups can form any suitable probe, tag, and / or label used for detection, which can be a molecular probe, labeled probe, hybridization. Includes probes, antibody probes, protein probes (eg, biotin binding probes), enzyme labels, fluorescent probes, fluorescent tags, and / or enzyme reporters.

Although this disclosure refers to luminescent markers, other types of markers may be used with the devices, systems, and methods provided herein. Such markers can be mass tags, electrostatic tags, or electrochemical labels.

Extrinsic markers can be added to the sample, but endogenous markers can already be part of the sample. The endogenous marker can include any luminescent marker present that can luminescence or "autofluorescent" in the presence of excitation energy. Autofluorescence of endogenous fluorophores can provide label-free and non-invasive labeling without the need for introduction of exogenous fluorophores. Examples of such endogenous fluorophores include, but are not limited to, hemoglobin, oxygen hemoglobin, lipids, collagen and elastin crosslinks, reduced nicotinamide adenine dinucleotide (NADH), oxidation. It is possible to include flavins (FAD and FMN), lipofustin, keratin, and / or porphyrins.

Although some embodiments may relate to diagnostic testing by detecting a single molecule in a sample, the present inventors have the ability to detect a single molecule in the present disclosure, eg, one or more of a gene. Recognized that it can be used to perform polypeptide (eg, protein) sequencing or nucleic acid (eg, DNA, RNA) sequencing of nucleic acid segments. Nucleic acid sequencing techniques vary in the methods used to determine nucleic acid sequences and can also vary in rates, read lengths, and error occurrences during the sequencing process. For example, some nucleic acid sequencing methods are based on synthetic sequencing, in which when a nucleotide is integrated into a newly synthesized strand of nucleic acid complementary to the target nucleic acid molecule. , The identity of the nucleotide is determined.

During sequencing, the polymerizing enzyme can be coupled (eg, attached) to the priming site of the target nucleic acid molecule. The priming site can be a primer complementary to the target nucleic acid molecule. As an alternative, the priming location is a gap or nick provided within the double-stranded segment of the target nucleic acid molecule. The gap or nick can be from 0 to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, or 40 nucleotides in length. Nick can provide cleavage within one strand of a double-stranded sequence, which can provide a priming site for a polymerizable enzyme, such as, for example, a chain-substituted polymerase enzyme. be.

In some cases, the sequencing primer may be annealed to the target nucleic acid molecule, whether or not the target nucleic acid molecule is immobilized on a solid support such as a sample well. good. In some embodiments, the sequencing primer may be immobilized on a solid support, and hybridization of the target nucleic acid molecule also immobilizes the target nucleic acid molecule on the solid support. Nucleotides can be added to the primer in a 5'-3'template bound fashion through the action of an enzyme (eg, polymerase) that can add or incorporate the nucleotide into the primer. Such integration of nucleotides into the primer (eg, via the action of a polymerase) can be commonly referred to as a primer extension reaction. Each nucleotide can be associated with a detectable tag, which determines the sequence of each nucleotide that is detected and used and incorporated into the primer, and thus the newly synthesized nucleic acid molecule. It is possible. The sequence of the target nucleic acid molecule can also be determined through the sequence complementarity of the newly synthesized nucleic acid molecule. In some cases, the annealing of the sequencing primer to the target nucleic acid molecule and the incorporation of the nucleotide into the sequencing primer are under similar reaction conditions (eg, the same or similar reaction temperature) or different. It can occur under reaction conditions (eg, different reaction temperatures). Moreover, some sequencing by synthetic methods may include the presence of a population of target nucleic acid molecules (eg, a copy of the target nucleic acid) and / or the step of amplifying the target nucleic acid to achieve the target nucleic acid population. It is possible.

According to embodiments, at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99. 99%, 99.999%, or 99.9999% accuracy and / or about 10 base pairs (bp), 50 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 1000 bp, 10,000 bp, 20,000 bp, Single nucleic acid molecules can be sequenced with high accuracy and long read lengths, such as 30,000 bp, 40,000 bp, 50,000 bp, or more than 100,000 bp read lengths. In some embodiments, the target nucleic acid molecule used in single molecule sequencing is a single-stranded target nucleic acid (eg, deoxyribonucleic acid (DNA), DNA derivative, ribonucleic acid (RNA), RNA derivative) template. Yes, it is added or immobilized in the sample well, and the sample well is immobilized or attached to a solid support such as the bottom of the sample well, a sequencing reaction (eg, DNA). Contains at least one additional component of a polymerase, such as a polymerase, a sequencing primer, etc.). The target nucleic acid molecule or polymerase can be attached to the sample wall, for example, directly at the bottom of the sample well or through a linker. The sample well can also contain any other reagent required for nucleic acid synthesis via the primer extension reaction, such as suitable buffers, cofactors, enzymes (eg, polymerases), etc. And, for example, deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyuridine triphosphate (dUTP), and deoxycytidine triphosphate (dTTP), Deoxyribonucleoside polyphosphates and the like, such as deoxyribonucleoside triphosphates, including dNTPs, which include luminescent tags such as fluorophores and the like. Each class of dNTPs (eg, dNTPs containing adenine (eg dATP), dNTPs containing cytosine (eg dCTP), dNTPs containing guanine (eg dGTP), dNTPs containing uracil (eg dUTP)). , And dNTPs containing thymine (eg, dTTP) are conjugated to a separate luminescent tag, and detection of light emitted from the tag is integrated into the newly synthesized nucleic acid. It is designed to show the identity of. The light emitted from the luminescent tag may be detected via any suitable device and / or method, including such devices and methods for detection described elsewhere herein. , It is possible (and therefore associated with dNTP) due to its suitable luminescent tag. The luminescent tag can be conjugated to dNTP at any position so that the presence of the luminescent tag does not block the integration of dNTP into the newly synthesized nucleic acid chain or the activity of the polymerase. .. In some embodiments, the luminescent tag is conjugated to a terminal phosphate (gamma phosphate) of dNTPs.

The single-stranded target nucleic acid template can be contacted with sequencing primers, dNTPs, polymerases, and other reagents required for nucleic acid synthesis. In some embodiments, all suitable dNTPs can be contacted simultaneously with a single-stranded target nucleic acid template (eg, all dNTPs are present simultaneously) so that integration of dNTPs can occur continuously. It has become. In other embodiments, the dNTPs may be sequentially contacted with a single-stranded target nucleic acid template, in which case the single-stranded target nucleic acid template is separately contacted with each suitable dNTP and single-stranded. A washing step is involved between the contact of the target nucleic acid template with different dNTPs. Such a cycle in which the single-stranded target nucleic acid template contacts each dNTP separately is repeated for each successive base position of the single-stranded target nucleic acid template to be followed and identified by washing. It can be.

Sequencing primers are annealed to the single-stranded target nucleic acid template, and the polymerase is continuous with dNTP (or other deoxyribonucleoside polyphosphoric acid) via the single-stranded target nucleic acid template. Incorporate. The unique luminescent tag associated with each integrated dNTP can be excited by the appropriate excitation light during or after incorporation of the dNTP into the primer, the emissions of which are described elsewhere herein. It can be subsequently detected using any suitable device and / or method, including devices and methods for detection. The detection of a particular emission of light can be attributed to a particular dNTP incorporated. Sequences obtained from the collection of detected luminescent tags can then be used to sequence single-stranded target nucleic acid templates via sequence complementarity.

Although this disclosure refers to dNTPs, the devices, systems, and methods provided herein include ribonucleotides and deoxynucleotides (eg, at least 4, 5, 6, 7, 8, 9, or 10). It can be used with various types of nucleotides, such as deoxyribonucleoside polyphosphate) with a phosphate group. Such ribonucleotides and deoxynucleotides can include various types of tags (or markers) and linkers.

The signal emitted during nucleoside integration can be stored in memory and processed at a later point in time to sequence the target nucleic acid template. This can include comparing the signal to the reference signal and determining the identity of the incorporated nucleoside as a function of time. Alternatively or in addition, the signals emitted during nucleoside integration can be collected and processed in real time (ie, during nucleoside integration) to sequence the target nucleic acid template in real time. ..

Nucleic acid sequencing of multiple single-stranded target nucleic acid templates can be completed if multiple sample wells are available, as in the case of the devices described elsewhere herein. Each sample well may be provided with a single-stranded target nucleic acid template and the sequencing reaction may be completed within each sample well. Each of the sample wells can be contacted with the appropriate reagents required for nucleic acid synthesis during the primer extension reaction (eg, dNTPs, sequencing primers, polymerases, cofactors, suitable buffers, etc.) and the sequencing reaction can be performed. , It is possible to proceed within each sample well. In some embodiments, the plurality of sample wells are contacted with all suitable dNTPs simultaneously. In another embodiment, the plurality of sample wells are separately contacted with each suitable dNTP and washed between contacts with different dNTPs. The integrated dNTPs can be detected in each sample well and the sequence can be determined for the single-stranded target nucleic acid in each sample well as described above.

Embodiments relating to single molecule nucleic acid sequencing can use any polymerase capable of synthesizing nucleic acids complementary to the target nucleic acid molecule. Examples of polymerases are not limited to these, but are limited to DNA polymerases, RNA polymerases, heat resistant polymerases, wild type polymerases, denatured polymerases, Escherichia coli DNA polymerases I, T7 DNA polymerases, Bacterophage T4 DNA polymerase φ29 (Pusai 29) DNA polymerases, Taq Polymerase, Tth Polymerase, Tli Polymerase, Pfu Polymerase, Pwo Polymerase, VENT Polymerase, DEEPVENT Polymerase, EX-Taq Polymerase, LA-Taq Polymerase, Sso Polymerase, Poc Polymerase, Pab Polymerase, Mth Polymerase, ES4 Polymerase, Tru Polymerase, Tac Polymerase, Tne Polymerase, Tma Polymerase, Tca Polymerase, Tih Polymerase, Tfi Polymerase, Platinum Taq Polymerase, Tbr Polymerase, Tfl Polymerase, Tth Polymerase, Pfutubo Polymerase, Pyrobest Polymerase, Pwo Polymerase, KOD Polymerase, Bst Polymerase, Sac Polymerase, 3' Includes Klenow fragment polymerases with exonuclease activity from to 5', and variants, modified products, and derivatives thereof. In some embodiments, the polymerase is a single subunit polymerase. In some embodiments, the polymerase is a highly progressive polymerase. Polymerase progression generally refers to the ability of polymerases to continuously integrate dNTPs into a nucleic acid template without releasing the nucleic acid template. During base pairing between the nucleobase of the target nucleic acid and the complementary dNTP, the polymerase creates a phosphodiester bond between the 3'hydroxyl end of the newly synthesized strand and the alpha phosphate of dNTP. By forming, dNTPs are incorporated into the newly synthesized nucleic acid chain. In the example where the luminescent tag conjugated to dNTP is a fluorophore, its presence is signaled by excitation and a pulse of emission is detected during or after the integration process. With respect to the detection label conjugated to the terminal (gamma) phosphate of dNTP, the incorporation of dNTP into the newly synthesized chain results in the release of beta phosphate and gamma phosphate, the detection label is the sample. -Can diffuse freely into the well, resulting in a reduction in emissions detected in the fluorophore.

Embodiments relating to single molecule RNA sequencing can use any reverse transcriptase capable of synthesizing complementary DNA (cDNA) from an RNA template. In such embodiments, the reverse transcriptase functions in a manner similar to that of a polymerase in that the cDNA can be synthesized from the RNA template via integration of dNTP into the reverse transcription primer annealed to the RNA template. It is possible to do. The cDNA can then be involved in the sequencing reaction and its sequence is determined as described above. The determined cDNA sequence is then used via sequence complementarity and it is possible to sequence the original RNA template. Examples of reverse transcriptase are Moloney Mouse Leukemia Virus Reverse Transcriptase (M-MLV), Chicken Myeluloblastosis Virus (AMV) Reverse Transcriptase, Human Immunodeficiency Virus Reverse Transcriptase (HIV-1), and Telomerase Reversal. Includes transcriptase.

Recognizing the need for a simpler and less complex device for performing single molecule detection and / or nucleic acid sequencing, we used a set of tags to detect single molecules and differ. I came up with a technique for labeling molecules. Such a single molecule can be a tagged nucleotide or amino acid. The tag can be detected during release from a single molecule while being attached to a single molecule, or upon release from a single molecule and attached to a single molecule. In some examples, the tag is a luminescent tag. Each luminescent tag in the selected set is associated with each molecule. For example, a set of four tags can be used to "label" a nucleobase present in DNA, and each tag in the set is associated with a different nucleobase, eg, the first tag. Is associated with adenine (A), a second tag is associated with cytosine (C), a third tag is associated with guanine (G), and a fourth tag is associated with guanine (G). It is associated with thymine (T). Moreover, each of the luminescent tags in the set of tags has different properties, which can be used to distinguish the first tag of the set from the other tags in the set. Thus, each tag can be uniquely identified using one or more of these distinguishing properties. The properties of tags that can be used to distinguish one tag from another, as an example and not as a limitation, are the emission energy and / or wavelength of light emitted by the tag in response to excitation. And / or it is possible to place the tag in an excited state, including the energy and / or wavelength of the excitation light that excites a particular tag.

Embodiments can use any suitable combination of tag properties to distinguish the first tag in a set of tags from other tags in the same set. For example, in some embodiments, it is possible to identify the tag using only the wavelength of the emission light from the tag. In such an embodiment, each tag in the selected set of tags has a different peak emission wavelength than the other tags in the set, and the luminescent tags are all simple. Excited by light from one excitation source. FIG. 1-1 illustrates emission spectra from four luminescent tags according to embodiments, where the four tags show their respective intensity peaks at different emission wavelengths, which are described herein. It is called the "peak emission wavelength" of the tag. The first emission spectrum 1-101 from the first luminescent tag has a peak emission wavelength at λ1 and the second emission spectrum 1-102 from the second luminescent tag has a second emission spectrum 1-102. The third emission spectrum 1-103 from the third luminescent tag has a peak emission wavelength at λ2 and has a peak emission wavelength at λ3 and is from the fourth luminescent tag. The fourth emission spectrum 1-104 of No. 1 has a peak emission wavelength at λ4. In this embodiment, the emission peaks of the four luminescent tags can have any suitable value that satisfies the relational expression λ1 <λ2 <λ3 <λ4. The four emission spectra may or may not overlap. However, if the emission spectra of two or more tags overlap, one tag emits substantially more light at each peak wavelength than any other tag. It is desirable to select a set. In this embodiment, the excitation wavelengths at which each of the four tags absorbs the light from the excitation source to the maximum are substantially the same, but not necessarily. Using the above tag set, four different molecules can be labeled with each tag from the tag set, the tag can be excited using a single excitation source, and the tag can be They can be distinguished from each other by detecting the emission wavelengths of the tags using optical systems and sensors. Although FIG. 1-1 illustrates four different tags, it should be recognized that any suitable number of tags can be used.

In other embodiments, it is possible to identify the tag using both the wavelength of the emission light from the tag and the wavelength at which the tag absorbs the excitation light. In such an embodiment, each tag in the selected set of tags has a different combination of emission wavelengths and excitation wavelengths from the other tags in the set. Thus, some tags in the selected tag set can have the same emission wavelength, but can be excited by light of different wavelengths. Conversely, some tags in the selected tag set can have the same excitation wavelength, but can emit light at different wavelengths. FIG. 1-2A illustrates emission spectra from four luminescent tags according to embodiments, two of which have a first peak emission wavelength and the other two tags. Has a second peak emission wavelength. The first emission spectrum 1-105 from the first luminescent tag has a peak emission wavelength at λ1 and the second emission spectrum 1-106 from the second luminescent tag also has a peak emission wavelength. The third emission spectrum 1-107 from the third luminescent tag has a peak emission wavelength at λ1 and has a peak emission wavelength at λ2 and is from the fourth luminescent tag. The fourth emission spectrum 1-108 of the above also has a peak emission wavelength at λ2. In this embodiment, the emission peaks of the four luminescent tags can have any suitable value that satisfies the relational expression λ1 <λ2. FIG. 1-2B illustrates the absorption spectra from the four luminescent tags, two of which have a first peak absorption wavelength and the other two tags have a second. It has a peak absorption wavelength. The first absorption spectrum 1-109 for the first light emitting tag has a peak absorption wavelength at λ3, and the second absorption spectrum 1-110 for the second light emitting tag has a peak absorption wavelength at λ4. The third absorption spectrum 1-111 with respect to the third light emitting tag has a peak absorption wavelength at λ3, and the fourth absorption spectrum 1-112 with respect to the fourth light emitting tag has. , Λ4 has a peak absorption wavelength. It should be noted that the tags that share the emission peak wavelength in FIG. 1-2A do not share the absorption peak wavelength in FIG. 1-2B. Using such a tag set makes it possible to distinguish between the four tags, even when there are only two emission wavelengths for the four dyes. This is possible by using two excitation sources that emit at different wavelengths, or a single excitation source that can emit at multiple wavelengths. If the wavelength of the excitation light is known for each detected emission event, it can be determined which tag was present. The excitation source can repeat the first and second excitation wavelengths, which is referred to as interleaving. Alternatively, it is possible that two or more pulses of the first excitation wavelength are used, followed by two or more pulses of the second excitation wavelength.

Although not shown in the figure, other embodiments are capable of determining the identity of the luminescent tag based solely on the absorption frequency. Such an embodiment is possible if the excitation light can be tuned to a particular wavelength that matches the absorption spectrum of the tag in the tag set. In such embodiments, the optical systems and sensors used to direct and detect the light emitted from each tag need not be able to detect the wavelength of the emitted light. This may be advantageous in some embodiments. The reason is that such embodiments do not require detection of emission wavelengths, which reduces the complexity of optical systems and sensors.

As discussed above, the inventor recognizes and understands the need to be able to distinguish different, optically (eg, luminescent) tags from each other using different properties of the tag. did. The type of property used to determine the identity of the tag influences the physical device used to perform this analysis. The present application discloses several embodiments of devices, devices, devices, and methods for performing these different experiments.

In short, we have identified multiple pixelated sensor devices in parallel with a relatively large number of pixels (eg, hundreds, thousands, millions, or more). Recognized and understood that it enables the detection of individual molecules or particles. Such a single molecule can be a tagged nucleotide or amino acid. The tag can be detected during release from a single molecule while being attached to a single molecule, or upon release from a single molecule and attached to a single molecule. In some examples, the tag is a luminescent tag. Molecules can be, by way of example, and without limitation, proteins and / or nucleic acids (eg, DNA, RNA). What's more, high-speed devices capable of acquiring data at over 100 frames per second enable detection and analysis of dynamic processes or changes that occur over time in the sample being analyzed.

We use a low-cost, single-use, single-use assay chip in connection with equipment including excitation light sources, optics, and optical sensors to emit optical signals from biological samples. Recognized and understood that it is possible to measure (eg, luminescent light). Using a low cost assay chip reduces the cost of performing a given bioassay. The biological sample is placed on the assay chip and can be discarded once a single bioassay is complete. In some embodiments, two or more types of samples can be analyzed simultaneously in parallel by placing multiple samples simultaneously on different parts of the assay chip. The assay chip interfaces with more expensive multi-use equipment, and the more expensive multi-use equipment can be used repeatedly with many different disposable assay chips. Low-cost assay chips that interface to compact portable devices can be used anywhere in the world without the constraints of high-cost biological laboratories that require laboratory expertise to analyze samples. obtain. Therefore, it is possible to bring automated biological analysis to areas of the world where previously quantitative analysis of biological samples was not possible. For example, by placing a blood sample on a disposable assay chip, by placing the disposable assay chip in a small portable device for analysis, and for immediate review by the user. Blood tests for young children can be performed by processing the results by a computer connected to the device. Data can also be transmitted to remote locations through the data network for analysis and / or archived for subsequent clinical analysis. Alternatively, the device can include one or more processors for analyzing the data obtained from the device's sensors.

Various embodiments are described in more detail below.
II. Overview of the Device by Some Embodiments A schematic overview of the device 2-100 is illustrated in FIG. 2-1. The system consists of both assay chip 2-110 and instrument 2-120, which consists of an excitation source 2-121 and at least one sensor 2-122. Assay chip 2-110 is interfaced to instrument 2-120 using any suitable assay chip interface. For example, the assay chip interface of instrument 2-120 to accept assay chip 2-110 and to a state of accurate optical alignment with the excitation source 2-110 and at least one sensor 2-122. It is possible to include a socket (not shown) to hold the assay chip 2-110. The external excitation source 2-121 in instrument 2-120 provides excitation energy to assay chip 2-110 for the purpose of exciting the sample in sample well 2-111 of assay chip 2-110. It is configured to provide to. In some embodiments, assay chip 2-110 has multiple pixels, with sample well 2-111 for each pixel accepting samples used in analysis independent of other pixels. It is configured as follows. Each pixel of assay chip 2-110 consists of a sample well 2-211 for receiving, holding, and analyzing a sample from the sample being analyzed. Such pixels can be referred to as "passive source pixels" because the pixel receives the excitation energy from an excitation source that is distant from the pixel. In some embodiments, there are pixels in instrument 2-120 that correspond to each pixel present on assay chip 2-110. Each pixel of instrument 2-120 is at least one sensor for detecting the emission energy emitted by the sample in response to the sample being illuminated by the excitation energy from the excitation source 2-121. Consists of. In some embodiments, each sensor can include multiple sub-sensors, each sub-sensor being configured to detect different wavelengths of emission energy from the sample. .. Two or more sub-sensors are capable of detecting emission energies of a particular wavelength, but each sub-sensor is capable of detecting different wavelength bands of emission energy.

In some embodiments, an optical element for guiding and coupling the excitation energy from the excitation source 2-121 to the sample well 2-111 is represented by arrow 2-101 in FIG. 2-1. As such, it is positioned on both the assay chip 2-110 and the instrument 2-120. Such a source-to-well element is positioned on the instrument 2-120 to couple the excitation energy to the assay chip 2-110, mirrors, lenses, dielectric coatings, and beams. It is possible to include a combiner and a lens, plasmonic element on the assay chip 1-110 to direct the excitation energy received from instrument 2-120 to the sample well 2-111. , And dielectric coatings can be included. Additionally, in some embodiments, an optical element for guiding the emission energy from sample well 2-111 to sensor 2-122 is represented by arrow 2-102 in FIG. 2-1. As such, it is located on the assay chip 2-110 and instrument 2-120. Such a well-to-sample element is positioned on the assay chip 2-110 to direct the emission energy from the assay chip 2-110 to the assay chip 2-110, the lens. It is possible to include a plasmonic element, and a dielectric coating, and on equipment 1-120 to direct the emission energy received from assay chip 2-110 to sensor 2-111, It is possible to include lenses, mirrors, dielectric coatings, filters, and diffractive optics. In some embodiments, a single component can play a role both in coupling the excitation energy to the sample well and in delivering the emission energy from the sample well to the sensor. It is possible.

In some embodiments, the assay chip 2-110 consists of multiple pixels, each pixel being its own individual sample well 2-111, and its own on instrument 2-120. It is associated with the associated sensor 2-122. Multiple pixels can be arranged in an array and can have any suitable number of pixels. For example, an assay chip has approximately 1,000 pixels, 10,000 pixels, approximately 100,000 pixels, approximately 1,000,000 pixels, approximately 1,000,000 pixels, Alternatively, it can contain approximately 100,000,000 pixels.

In some embodiments, the device 2-120 comprises a sensor chip consisting of a plurality of sensors 2-122, the plurality of sensors 2-122 being arranged as a plurality of pixels. Each pixel of the sensor chip corresponds to a pixel in assay chip 2-110. Multiple pixels can be arranged in an array and can have any suitable number of pixels. In some embodiments, the sensor chip has the same number of pixels as assay chip 2-110. For example, a sensor chip may have approximately 10,000 pixels, approximately 100,000 pixels, approximately 1,000,000 pixels, approximately 10,000,000 pixels, or approximately 100,000 pixels. It is possible to include 000 pixels.

Instrument 2-120 is interfaced to assay chip 2-110 through an assay chip interface (not shown). The assay chip interface includes components for positioning and / or aligning assay chip 2-110 with respect to instrument 2-120, and assay chip 2-110 the excitation energy from excitation source 2-121. It is possible to improve the coupling to. In some embodiments, the excitation source 2-121 comprises a plurality of excitation sources combined to deliver the excitation energy to the assay chip 2-110. Multiple excitation sources can be configured to produce multiple excitation energies, which correspond to light of different wavelengths.

Device 2-120 includes a user interface 2-125 for controlling the operation of the device. User interface 2-125 is configured to allow the user to enter information such as commands and / or settings used to control the functionality of the device into the device. .. In some embodiments, the user interface 2-125 can include a microphone for buttons, switches, dials, and voice commands. In addition, user interface 2-125 is about proper alignment and / or performance of the instrument and / or assay chip, such as information obtained by a read signal from the sensor on the sensor chip. Feedback can be made available to the user. In some embodiments, the user interface 2-125 uses a speaker to provide audible feedback and / or an indicator light and / or display screen to provide visual feedback. It is possible to provide feedback. In some embodiments, the device 2-120 comprises a computer interface 2-124 used to connect to the computing device 2-130. Any suitable computer interface 2-124 and computing device 2-130 may be used. For example, computer interface 2-124 can be a USB interface or a firewire ™ interface. The computing device 2-130 can be any general purpose computer such as a laptop computer, desktop computer, or tablet computer, or a mobile device such as a mobile phone. .. Computer interface 2-124 facilitates the communication of information between device 2-120 and computing device 2-130. Input information for controlling and / or configuring device 2-120 may be provided through computing device 2-130 connected to device computer interface 2-124. Additionally, the output information may be received by computing device 2-130 through computer interface 2-124. Such output information can include feedback on the performance of device 2-120 and information from the read signal of sensor 2-122. Equipment 2-120 can also include processing devices 2-123 for analyzing data received from sensors 2-122. In some embodiments, the processing device 2-123 may be a general purpose processor (eg, a central processing unit (CPU), a field programmable gate array (FPGA), or an application specific integrated circuit (ASIC)). It is possible to have a custom integrated circuit such as). In some embodiments, processing the data from sensor 2-122 can be done by both processing device 2-123 and external computing device 2-130. In other embodiments, computing device 2-130 may be omitted, and processing data from sensors 2-122 may be performed solely by processing device 2-123.

When the excitation source 2-121 irradiates the assay chip 2-110 with excitation energy, the sample in one or more pixels of the assay chip 2-110 can be excited. In some embodiments, the sample is labeled with a plurality of markers, each associated with a different sample within the sample and identifiable by emission energy. The path from sample well 2-111 to sensor 2-122 can include one or more components that help identify multiple markers based on emission energy. The component is capable of focusing emission energies towards sensor 2-122 and, additionally or alternatively, spatially separates emission energies with different characteristic energies and thus different wavelengths. It is possible to do. In some embodiments, the assay chip 2-110 can include a component that directs the emission energy towards the sensor 2-122, and the device 2-120 has a different wavelength of emission energy. It is possible to include a component for spatially separating the. For example, optical filters or diffractive optics can be used to couple the wavelengths of emission energy to spatial degrees of freedom. The sensor or sensor area can contain a plurality of sub-sensors, the plurality of sub-sensors being configured to detect the spatial distribution of emission energy depending on the radiation pattern. Luminescent tags that emit different emission energies and / or spectral ranges can form different radiation patterns. The sensor or sensor area can detect information about the spatial distribution of emission energy that can be used to identify markers among multiple markers.

Emission energy from the sample in sample well 2-110 can be detected by sensors 2-122 and converted into at least one electrical signal. The electrical signal is transmitted along a conductive line in the circuit of equipment 2-120 and can be processed and / or analyzed by processing device 2-123 and / or computing device 2-130.

FIG. 2-2 is a top view of the assay chip 2-110 and a top view of the sensor chip 2-260, illustrating the correspondence between the pixels of the two chips. Assay chip 2-110 consists of multiple pixels, each pixel containing sample well 2-111 formed in conductive material 2-221. The sensor chip 2-260 is also composed of a plurality of pixels, each pixel including a sensor 2-121 formed in or on the substrate 2-247. The arrows in FIG. 2-2 illustrate the correspondence between two of the pixels of assay chip 2-110 and two of the pixels of sensor chip 2-260. Although not shown for clarity, each pixel of assay chip 2-110 is associated with a pixel of sensor chip 2-260.

An overview of several components associated with a single pixel on assay chip 2-110 and a single pixel on sensor chip 2-260 is illustrated in Figure 2-3. Instrument 2-100 consists of both assay chip 2-110 and instrument 2-120. In some embodiments, assay chip 2-110 is a disposable chip designed for analysis of a single sample. Assay chip 2-110 comprises one or more metal layers 2-221, one or more dielectric layers 2-225, and focusing element 2-227. In some embodiments, the metal layer 2-221 comprises a stack of layers, some of which may include an absorbent layer. Instrument 2-120 includes one or more excitation sources 2-250, at least one polychromic mirror 2-230, and sensor chip 2-260, where sensor chip 2-260 contains filtering element 2. It is possible to include -241, a spectral sorting element 2-243, a focusing element 2-245, and at least one sensor 2-122 in or on substrate 2-247. FIG. 2-3 illustrates only a single pixel on assay chip 2-110 and only a single pixel on sensor chip 2-260, but with excitation source 2-250, polychromic mirror 2. Some components of equipment 2-120, such as -230, and filtering elements 2-241, can be common to multiple pixels. For example, in some embodiments, a single excitation source 2-250 and polychromic mirror 2-230 are capable of directing excitation energy to all pixels of assay chip 2-110.

In some embodiments, the sample can include body fluids such as blood, urine, or saliva. The sample wells 2-211, in the metal layer 2-221, form a sample volume for the sample from the sample to enter. The opening at the end of sample well 2-211 may be referred to as a nanoaperture. The nanoaperture can have a width smaller than the wavelength of the excitation energy 2-251 emitted by the excitation source 2-250. A portion of a sample, referred to as a sample, can enter the sample volume defined by sample wells 2-211. The sample can be any particle, molecule, protein, genetic material, or any other sample present in the sample.

The excitation source 2-250 emits an excitation energy 2-251, which is directed towards the sample well 2221 and irradiates the sample. In some embodiments, the excitation source 2-251 can be a single light source that provides excitation energy for all pixels of assay chip 2-110. The polychromic mirror 2-230 reflects light from the excitation source 2-250 and directs the excitation energy 2-251 towards one or more sample wells 2-211 of assay chip 2-110. do. Therefore, in some embodiments, a single polychromic mirror that directs the excitation energy towards all sample wells, rather than having each pixel associated with its own polychromic mirror. Only 2-230 can be present. Similarly, there can be a one-to-many relationship between other optical elements used to direct the excitation energy towards the sample wells 2-211.

Concentric circular gratings 2-223 can be formed adjacent to the bottom nanoaperture of sample wells 2-211. The concentric circular gratings 2-223 can project from the bottom surface of the metal layer 2-221. The sample well 2-211 may be located at the center of the circular grating 2-223 or near the center of the circular grating 2-223. Both the sub-wavelength scale of the nanoaperture of sample well 2-211 and the concentric circular gratings 2-223 generate a field-enhancing effect, and the field-enhancing effect is in sample well 2-211. It increases the intensity of the excitation energy and results in an increase in the coupling of the excitation energy to the sample present in the sample wells 2-2111. At least from time to time, the sample absorbs photons from the excitation energy and emits photons with less energy than that of the excitation energy 2-251 (referred to as "emission energy" 2-253). Emission energy 2-253 can be emitted downwards. The circular grating 2-223 serves as a plasmonic element, which is used to reduce the spread of emission energies 2-253 and towards the associated sensor emission energies 2 -Can be used to direct 253.

Emission energy 2-253 travels through the dielectric layer 2-225, and the dielectric layer 2-225 is used to allow the emission energy 2-253 to propagate some distance. It can be a spacer layer. The dielectric layer 2-225 is also capable of providing structural strength to assay chip 2-110. Emission energy 2-253 then proceeds through one or more focusing elements 2-227, where one or more focusing elements 2-227 are sensor chips 2-2260 in equipment 2-120. Used to further direct emissions energy 2-253 to sensors 2-122 in the relevant pixels of.

The polychromic mirror 2-230 then passes through the emission energy 2-253 and reflects a portion of any excitation energy 2-251 reflected from the assay chip 2-110. A portion of the excitation light that is not reflected by the assay chip 2-110 is either transmitted through the assay chip or absorbed by the assay chip. To further reduce the amount of excitation energy 2-251 reflected by assay chip 2-110 and not reflected by polychromic mirror 2-230, filtering element 2-241 optics towards sensor chip 2-260. Can be arranged in a specific path. Filtering elements 2-241 can include, by way of example, and without limitation, broadband filters, notch filters, or edge filters, which transmit emission energy 2-253. Absorbs and / or reflects excitation energy 2-251.

In embodiments, spectral sorting elements 2-243 are used to facilitate the determination of the identity of the markers in sample wells 2-211, using the spectral properties of emission energy 2-253. Is included on the sensor chip 2-260 and is capable of coupling the spectral degrees of freedom of emission energy 2-253 in the direction in which emission energy 2-253 is advancing. For example, diffractive optics are used to direct the emission energy 2-253 of the first wavelength in the first direction and the emission energy 2-253 of the second wavelength in the second direction. It is possible. One or more focusing elements 2-245 may be used to direct spectrally sorted light onto sensors 2-122. Sensors 2-122 can include one or more sub-sensors (not shown), each for redirection of light of different wavelengths by spectral sorting elements 2-243. Based on, it is associated with different wavelengths of emission energy 2-253.

The above description of FIG. 2-3 is an overview of some (but not all) of the components of the device according to some embodiments. In some embodiments, the one or more elements of FIG. 2-3 may be absent or may be in different locations. The components of Assay Chip 2-210 and Instrument 2-220 are described in more detail below.

Assay chips 2-110 and instrument 2-120 may be mechanically matched, detachably coupled, and separable from each other. The device 2-120 can include the device housing, and the mounting board 2-405 is disposed inside the device housing. FIG. 2-4 illustrates at least some of the components that may be included on the mounting board 2-405 of equipment 2-120. The mounting board 2-405 can include a printed circuit board, the mounting board 2-405 is a sensor chip 2-260 (not visible in Figure 2-4), heat sink 2-407, And it is possible to have an optical housing 2-401 mounted on it. Various optical components of equipment 2-120 may be disposed within the optical housing 2-401. In some embodiments, the equipment housing and mounting board can be of any suitable size. For example, the mounting board can be substantially circular with a diameter of 17.78 to 20.32 cm (7 to 8 ").

Assay chip 2-110 is coupled to optical housing 2-401 to ensure alignment with the optical components within optical housing 2-401. The chip holder frame 3-102 may be aligned with the opening of the optical housing 2-401. Preferably, the assay chip 2-110 can be detachably coupled to instrument 2-120. Any suitablely shaped magnetic component 2-403a-2-403b, such as a magnetic cylinder, may be installed around the opening of the optical housing 3-401 to open the opening of the optical housing 3-401. Through, the excitation energy exits the optical housing 2-401. Additionally, the magnetic components 2-403a to 2-403c can be calibrated to keep the chip holder frame 3-102 aligned with the opening. The chip holder frame can be positioned with micron level accuracy using an alignment cylinder. In some embodiments, three magnetic cylinders 2-403a to 2-403b are used to generate chip holder frame alignment. However, embodiments are not so limited and any suitable number of magnetic, spring-loaded, pneumatic, or other such components are in the right place in a matched configuration. Can be used to hold chips in. For example, the chip holder frame 3-102 may be held in place by a non-magnetic element such as a spring, pneumatic pressure, or suction from vacuum. Optionally, the chip holder frame 3-102 can be constructed using any rigid material suitable for positioning the chip in alignment with the optical block.

According to some aspects of the application, the distance between the sample well and the sensor can be kept small when the chip is connected to the system. In some embodiments, the optical distance between the sample well and the sensor can be less than 30 cm, less than 10 cm, less than 15 cm, or less than 1 cm.

III. Assay Chip In some embodiments, the assay chip 2-110 does not include any active electronic component. Both the excitation source 2-250 and the sensor 2-122 for each pixel are located off-chip inside instrument 2-120.

In some embodiments, the assay chip 2-110 may be housed within a chip holder frame 3-102, as illustrated in FIG. 3-1A. The chip holder frame 3-102 can be disposable and can be disposed of with the assay chip 2-110 after a single use. Assay chips 2-110 can be located behind the chip holder frame 3-102, as illustrated in FIG. 3-1B. The chip holder frame 3-102 can be made of any suitable ferromagnetic metal such as steel, with magnetic components 2-403a to 2-403b secured to the optical housing 2-401. , Chip holder frame 3-102 and thus assay chip 2-110 are designed to be held in place. In some embodiments, the chip holder frame 3-102 may be attached to the upper surface of the optical housing 2-401, as illustrated in FIG. 2-4.

In another embodiment illustrated in FIG. 3-1C, the assay chip may be attached to the top surface of the chip holder frame 3-102. A plastic cap 3-103 surrounds the assay chip 2-110 so that the pixel array of the assay chip 2-110 is exposed through an opening in the plastic cap 3-103. It has become. The user of Assay Chip 2-110 can place the sample in the opening of the plastic cap 3-103. By contacting the top surface of Assay Chip 2-110, the sample in the sample can be introduced into one or more of the multiple pixels of Assay Chip 2-110 for analysis. .. In some embodiments, no fluid channel or device is required to deliver a portion of the sample to the pixel via forced fluid flow.

A. Sample Well Layer As shown in FIG. 2-3 and in more detail in FIG. 3-2, some embodiments are one of assay chips 2-110 or Includes sample wells 2-211, formed on multiple pixels. The sample well can consist of a small volume or region formed within the metal layer 2-221, where the sample is deposited on the surface of assay chip 2-110. It is arranged so that it can diffuse from the sample well into the sample well and out of the sample well. In various embodiments, the sample wells 2-211 may be arranged to receive excitation energy from the excitation source 2-250. The sample that diffuses into the sample well can be temporarily or permanently retained in the excited region 3-215 of the sample well by the adherent 3-2111. In the excited region, the sample can be excited by excitation energy (eg, excitation light 3-245) and then can release energy, which can be observed and evaluated to characterize the sample. be.

In further detail of operation, at least one sample 3-101 to be analyzed is introduced into sample wells 2-211, for example, from a sample containing a fluid suspension of the sample (not shown). obtain. Excitation energy 3-245 from the excitation source 2-250 of device 2-120 is capable of exciting a sample or at least one tag (also referred to as a biological marker, reporter, or probe). , At least one tag is attached to the sample, or is otherwise associated with the sample while it is in the excitation region 3-215 in the sample well. According to some embodiments, the tag can be a luminescent molecule (eg, a luminescent tag or probe) or a quantum dot. In some embodiments, it is possible that there are more than one tag used to analyze the sample (eg, from a single polymerase molecule by J. Eid et al.). Single-molecule gene sequencing as described in Real-Time DNA Sequencing from Single Polymerase Moleculars, Science 323, p. 133 (2009), which is incorporated herein by reference. Separate tag used for). The sample or tag is capable of releasing emission energy during and / or after excitation. When multiple tags are used, they can emit with different characteristic energies (and thus have different wavelengths) and / or with different temporal characteristics. Emissions from sample wells 2-211 can be radiated to sensors 2-122 of device 2-120, at sensor 3-260 they are detected and converted into electrical signals and the electrical signals are , Can be used to characterize the sample.

According to some embodiments, the sample well 2-211 can be a partially enclosed structure, as shown in FIG. 3-2. In some embodiments, the sample wells 2-211 are submicron-sized holes or openings (at least one transverse dimension D _sw ) formed in at least one layer of material 2-211. Characterized) consists of. According to some embodiments, the transverse dimensions of the sample wells can be between approximately 20 nanometers and approximately 1 micron, but in some embodiments larger sizes and more. Smaller sizes can be used. The volume of sample well ^2-211 , in some embodiments, can be between about ^10-21 liters and about 10-15 liters. The sample well can be formed as a waveguide, which can or cannot support a propagation mode. In some embodiments, the sample well is formed as a zero-mode waveguide (ZMW) having a cylindrical shape (or similar shape) with a diameter (or maximum transverse dimension) D _sw . Can be done. ZMW can be formed in a single metal layer as nanoscale holes, which do not support propagation optical modes through the holes.

Due to the small volume of sample wells 2-211, a single in each pixel, even if the sample can be concentrated in the sample to be inspected at a concentration similar to that found in the natural environment. It may be possible to detect sample events (eg, single molecule events). For example, the micromolarity of the sample can be present in the sample placed in contact with the assay chip, but at the pixel level. The sample wells of Assay 2-110 are statistically sized to contain only about one sample, which is likely to be sample-free for single molecule analysis. Can be done. For example, in some embodiments, 30-40% of the sample wells contain a single sample. However, the sample well may contain more than one sample. Since a single molecule or a single sample event can be analyzed at each pixel, the assay chip detects unusual events that would otherwise go unnoticed in the ensemble average measurement. Make it possible.

The transverse dimension _Dsw of the sample well can be between about 500 nanometers (nm) and about 1 micron in some embodiments and from about 250 nm in some embodiments. It can be between about 500 nm, in some embodiments between about 100 nm and about 250 nm, and in some embodiments between about 20 nm and about 100 nm. It is possible to be. According to some embodiments, the transverse dimension of the sample well is between approximately 80 nm and approximately 180 nm, or approximately one-fourth to one-eighth of the excitation or emission wavelength. Is possible. According to other embodiments, the transverse dimensions of the sample wells are from about 120 nm to about 170 nm. In some embodiments, the depth or height of sample wells 2-211 can be between about 50 nm and about 500 nm. In some embodiments, the depth or height of sample wells 2-211 can be between about 80 nm and about 200 nm.

Sample wells 2-211, which have sub-wavelength transverse dimensions, can improve the behavior of pixel 2-100 on assay chip 2-110 in at least two ways. For example, the excitation energy 3-245 incident on the sample well from the opposite side of the sample can be coupled into the excitation region 3-215 with an exponentially decreasing power, and the sample well. It does not propagate through to the sample. As a result, the excitation energy is increased in the excitation region where it excites the sample of interest and is also reduced in the sample where it could excite other samples that would contribute to background noise. .. Also, emissions from the sample held at the base of the well are preferably oriented towards the sensor on device 2-120. The reason is that emissions cannot propagate upwards through the sample wells. Both of these effects are capable of improving the signal-to-noise ratio at the pixel. The inventor has recognized several aspects of the sample well that can be improved to further increase the signal-to-noise level in the pixel. These embodiments relate to the well shape and structure, and also the sample wells and adjacent optical and plasmonic structures that assist in coupling the energy emitted from the sample wells with the excitation energy. Regarding the relative position with (explained below).

According to some embodiments, the sample wells 2-211 can be formed as sub-cutoff nanoapertures (SCNs) that do not support propagation modes. For example, the sample well 2-211 can consist of a cylindrical hole or bore in the conductive layer 2-221. The cross section of the sample well does not have to be round and can be elliptical, square, rectangular, or polygonal in some embodiments. Excitation energy 3-245 (eg, visible or near-infrared radiation) can enter the sample well through entrance aperture 3-212, which is shown in FIG. 3-212. As shown in 2, at the first end of the well, it may be defined by the wall portion 3-214 of the sample well 2-211. When formed as an SCN, the excitation energy 3-245 can be exponentially attenuated along the SCN. In some embodiments, the waveguide can consist of an SCN for the energy emitted from the sample, but it does not have to be an SCN for the excitation energy. For example, the apertures and waveguides formed by the sample wells can be large enough to support propagation modes with respect to excitation energy. The reason is that it can have wavelengths shorter than the emitted energy. At longer wavelengths, emissions can exceed the cutoff wavelength for propagation modes in the waveguide. According to some embodiments, the sample well 2-211 can consist of an SCN with respect to the excitation energy 3-245, where the maximum intensity of the excitation energy is at the entrance to the sample well 2-211. It is designed to be localized to the excited region 3-215 of the sample well (eg, near the interface between layers 3-235 and 2-221, as shown in Figure 3-2). Localized to). Such localization of excitation energy can increase the emission energy density from the sample, and by confining the excitation energy in the vicinity of the entrance aperture 3-212, the emissions observed are single. It is possible to limit to a sample of (eg, a single molecule).

An example of excitation localization near the entrance of a sample well consisting of SCN is shown in Figure 3-3. Numerical simulations were performed to determine the intensity of excitation energies in and near sample wells 2-21 formed as SCNs. The results show that the intensity of the excitation radiation is about 70% of the incident energy at the entrance aperture of the sample well and drops to about 20% of the incident intensity within about 100 nm in the sample well. Shows. For this simulation, the characteristic wavelength of the excitation energy was 633 nm and the diameter of the sample wells 2-211 was 140 nm. Sample wells 2-211 were formed in a layer of gold metal. Each horizontal section in the graph is 50 nm. As shown by the graph, more than half of the excitation energy received in the sample wells is localized to about 50 nm in the entrance aperture 3-212 of the sample wells 2-211.

Other sample well structures have been developed and studied by the present inventor to improve the intensity of the excitation energy localized in sample well 2-211. FIG. 3-4 shows an embodiment of a sample well comprising a cavity or dibot 3-216 at the excited end of the sample well 2-211. As can be seen in the simulation results of FIG. 3-3, a region of higher excitation intensity exists just before the entrance aperture 2-212 of the sample well. According to some embodiments, the addition of Divot 3-216 to sample well 2-211 allows the sample to move into the region of higher excitation intensity. In some embodiments, the shape and structure of the divot is locally excited (eg, due to the difference in index of refraction between layer 3-235 and the fluid of the sample in the sample well). It is possible to change the field and further increase the intensity of the excitation energy in the dibot.

The dibot can have any suitable shape. The divot can have a transverse shape that is substantially uniform to the transverse shape of the sample well, for example round, elliptical, square, rectangular, polygonal. In some embodiments, the sidewalls of the divot can be substantially straight and vertical, similar to the walls of the sample wells. In some embodiments, the sidewalls of the divot can be tilted and / or curved, as shown in the drawings. The transverse dimensions of the divot can be approximately the same size as the transverse dimensions of the sample well in some embodiments, and in some embodiments the transverse dimensions of the sample well. It can be smaller than, or in some embodiments, larger than the transverse dimension of the sample well. The dibot 3-216 can extend beyond the metal layer 2-221 of the sample well at approximately 10 nm to approximately 200 nm. In some embodiments, the dibot can extend beyond the metal layer 2-221 of the sample well at approximately 50 nm to approximately 150 nm. By forming a dibot, the excited regions 3-215 can extend outside the metal layer 221 of the sample well, as shown in FIG. 3-4.

FIG. 3-5 shows the improvement of the excitation energy in the excitation region for the sample well containing the divot (shown in the simulation image on the left). For comparison, the excitation field is also simulated for sample wells without divot, which is shown on the right. The field size is converted from the color renderings in these plots, and the dark areas at the base of the divot represent higher intensities than the light areas in the sample wells. The dark area above the sample well represents the lowest intensity. As can be seen, the dibot allows sample 3-101 to move to the region of higher excitation intensity, and the dibot also localizes the region of highest intensity at the excited end of the sample well. To increase. Note that the high intensity regions are more dispersed with respect to the sample wells without divot. In some embodiments, the Divot 3-216 provides a two-fold or greater increase in excitation energy in the excited region. In some embodiments, a fold or more increase can be obtained depending on the shape and depth of the divot. In these simulations, the sample well consists of a layer with a thickness of 100 m, a divot with a depth of 50 nm, and an excitation energy at a wavelength of 635 nm.

FIG. 3-6 shows another embodiment of sample well 2-211, in which a sample well comprising a dibot is formed above the protrusion 3-615 on the surface of the substrate. There is. The resulting structure for the sample wells can increase the excitation energy in the sample more than twice as much as the sample wells shown in Figure 3-2, from the sample wells to the device 2. It is possible to direct the emissions to point at the -120 sensor. According to some embodiments, the protrusions 3-615 are patterned in a first layer 3-610 of the material. The overhangs can, in some embodiments, be formed as a circular pedestal or a ridge with a rectangular cross section, and a second layer 3-620 of material can be deposited above the first layer and the overhangs. .. At the overhang, the second layer is capable of forming a shape above the overhang that approximates the cylindrical portion 3-625, as shown. In some embodiments, a conductive layer 3-230 (eg, reflective metal) is deposited above the second layer 3-620, with sample wells 3-210 in the conductive layer above the protrusions. It can be patterned to form. The Divot 3-216 can then be etched into the second layer. The dibot can extend below the conductive layer 3-230 at about 50 nm to about 150 nm. According to some embodiments, the first layer 3-610 and the second layer 3-620 can be optically transparent and may or may be formed from the same material. It does not have to be done. In some embodiments, the first layer 3-610 may be formed from an oxide (eg, SiO ₂ ) or a nitride (eg, Si 3N ₄ ) and the second layer _3-620 may be oxidized. It can be formed from an object or a nitride.

According to some embodiments, the conductive layer 3-230 above the protrusion 3-625 is shaped as an approximately spherical reflector 3-630. The shape of the spherical portion can be controlled by the choice of protrusion height h, protrusion diameter or transverse dimension w, and thickness t of the second layer 3-620. The location of the excited region and the position of the sample can be adjusted with respect to the optical focus of the spherical reflector by selecting the divot depth d. The cylindrical reflector 3-630 is capable of concentrating the excitation energy in the excitation region 3-215 and also collects the energy emitted from the sample and reflects the radiation towards the sensor 3-260. It can be understood that it is possible to concentrate.

As mentioned above, the sample wells can be formed in any suitable shape and are not limited to cylindrical shapes. In some embodiments, the sample well can be a cone, tetrahedron, pentahedron, and the like. 3-7A-7F illustrate some exemplary sample well shapes and structures that may be used in some embodiments. Sample wells 2-211, according to some embodiments, may be formed to have a first aperture 2-212 that is greater than the second aperture 2-218 with respect to excitation energy. The sidewalls of the sample well can be tapered or curved. This formation of the sample well can allow more excitation energy to enter the excitation region and can further visibly attenuate the excitation energy traveling towards the sample. be. In addition, the emissions emitted by the sample can be preferentially emitted towards the end of the sample well with a larger aperture due to the preferred energy transfer in that direction.

In some embodiments, the Divot 3-216 is capable of having smaller transverse dimensions than the base of the sample well, as shown in Figure 3-7B. Smaller dibots can be formed by coating the sidewalls of the sample well with a sacrificial layer prior to etching the dibot and then removing the sacrificial layer. Smaller dibots can be formed to hold the sample in a region more equidistant from the conductive wall of the sample well. Holding the sample equidistant from the wall of the sample well has the undesired effect of the wall of the sample well on the radiating sample, eg, quenching the emissions and / or altering the radiation lifetime. Can be reduced.

3-7C and 3-7D show another embodiment of the sample well. According to this embodiment, the sample well 2-211 can consist of an excitation energy-enhanced structure 3-711 and an adhesive substance 3-221 formed adjacent to the excitation energy-enhanced structure. be. The energy-enhanced structure 3-711, according to some embodiments, consists of a surface plasmon or nano-antenna structure formed in a conductive material on an optically transparent layer 3-235. Is possible. FIG. 3-7C shows an elevation view of the sample wells 2-211, and a structure in the immediate vicinity, and FIG. 3-7D shows a plan view. The excitation energy enhancement structure 3-711 may be shaped and arranged to enhance the excitation energy within a small localized region. For example, the structure can include a pointed conductor with an acute angle in the sample well, which increases the intensity of the excitation energy in the excitation region 3-215. In the example shown, the excitation energy-enhanced structure 3-711 is in the form of a bow tie. Sample 3-101 diffused into the region is temporarily or permanently retained by the adhesive material 3-211, and also delivered from the excitation source 2-250 located within device 2-120. Can be excited by possible excitation energies. According to some embodiments, the excitation energy is capable of driving a surface plasmon current in the energy-enhanced structure 3-711. The resulting surface plasmon currents can create high electric fields at the sharply pointed tips of structure 3-711, which excite the sample held in the excitation region 3-215. It is possible to do. In some embodiments, the sample well 2-211, shown in FIG. 3-7C, can include a dibot 3-216.

Another embodiment of the sample well is shown in FIG. 3-7E, showing the excitation energy-enhanced structure 3-720 formed along the inner wall of the sample well 2-211. The excitation energy-enhanced structure 3-720 can consist of metal or conductor and can be formed using angled (or shadow) directional deposition, where sample wells are placed on it. The substrate formed is rotated during deposition. During deposition, the base of sample wells 2-211 is obscured by the upper sidewalls of the wells to prevent the deposited material from accumulating on the base. The resulting structure 3-720 is capable of forming an acute angle 3-722 at the bottom of the structure, and this acute angle of the conductor is capable of enhancing the excitation energy in the sample wells. ..

In the embodiment shown in FIG. 3-7E, the material 3-232 in which the sample well is formed does not have to be a conductor and is any suitable material such as a dielectric material. Is possible. According to some embodiments, the sample wells 2-211 and the excitation energy-enhanced structure 3-720 can be formed in bottomed holes etched into the dielectric layer 3-235, which are separate layers. 3-232 does not need to be deposited.

In some embodiments, shadow evaporation is then performed on top of the structure shown in Figure 3-7E, and at the base of the sample well, a metallic or conductive energy-enhanced structure. It is possible to deposit a body, eg, a trapezoidal structure or a pointed cone, as indicated by the dotted line. The energy-enhanced structure is capable of enhancing the excitation energy into the wells via surface plasmons. After shadow evaporation, a flattening process (eg, a chemical and mechanical polishing process or a plasma etching process) is performed, leaving the energy-enhancing structure in the well at the top of the sample well. It is possible to remove or etch back the deposited material.

In some embodiments, the sample wells 2-211 can be formed from more than a single metal layer. FIG. 3-7F illustrates sample wells formed within a multilayer structure, where different materials can be used for different layers. According to some embodiments, the sample well 2-211 is a first layer 3-232 (which can be a semiconductor or conductive material), a second layer 3-234 (which can be a semiconductor material or a conductive material). It can be an insulator or a dielectric), and can be formed in a third layer 2-221, which can be a conductor or a semiconductor. In some embodiments, degenerately doped semiconductors or graphene may be used for the layer of sample wells. In some embodiments, the sample wells can be used in two layers, in other embodiments the sample wells can be formed in four or more layers. In some embodiments, the multilayer material used to form the sample well is such that it increases surface plasmon generation at the base of the sample well or suppresses surface plasmon radiation at the top of the well. Can be selected. In some embodiments, the multilayer material used to form the sample wells may be selected to suppress the excitation energy propagating across the sample wells and the multilayer structure into the bulk sample.

In some embodiments, the multilayer material used to form the sample wells may be selected to increase or suppress the interfacial excitons that may be generated by the excitation energy incident on the sample wells. Multi-exciton, such as bi-exciton and tri-exciton, can be generated at the interface between two different semiconductor layers adjacent to the sample well. The sample well can be formed in both the metal layer and the first semiconductor layer, and the interface between the first semiconductor layer and the second semiconductor layer is located in the excited region 3-215 of the sample well. There is now. Interfacial excitons can have a longer lifetime than excitons in the volume of a single semiconductor layer, increasing the likelihood that excitons will excite samples or tags via FRET or DET. .. In some embodiments, at least one quantum dot from which the multi-exciton can be excited can be attached to the bottom of the sample well (eg, by a linking molecule). Also, excitons excited at quantum dots can have a longer lifetime than excitons in the volume of a single semiconductor layer. Interfacial excitons, or excitons generated at quantum dots, can increase the rate of FRET or DET, according to some embodiments.

Various materials can be used to form the sample wells described in the embodiments described above. According to some embodiments, the sample well 2-211 may be formed from at least one layer of material 3-230, which material 2-221 can be any of conductive materials, semiconductors, and insulators. It can consist of one or a combination thereof. In some embodiments, the sample wells 2-211 consist of a highly conductive metal layer, such as gold, silver, aluminum, copper. In some embodiments, layer 2-221 can consist of a multi-layer stack containing any one or a combination of gold, silver, aluminum, copper, titanium, titanium nitride, and chromium. .. In some embodiments, other metals may be used additionally or as an alternative. According to some embodiments, the sample wells can be made of an alloy, such as AlCu or AlSi.

In some embodiments, multiple layers of different metals or alloys can be used to form sample wells. In some embodiments, the material from which the sample wells 2-211 are formed can consist of alternating layers of metal and non-metal, such as alternating layers of metal and one or more dielectrics. Is. In some embodiments, the non-metal can include polymers such as polyvinyl phosphonic acid or polyethylene glycol (PEG) -thiol.

The layer 2-221 on which the sample well is formed is, according to some embodiments, on or at least one optically transparent layer 3-235 or at least one optically transparent layer. Can be deposited adjacent to layers 3-235, with little attenuation of excitation energies (eg, in visible or near-infrared form) and emission energies (eg, in visible or near-infrared form). It is now possible to proceed to and from sample wells 2-211, and from sample wells 3-210. For example, the excitation energy from the excitation source 2-250 can pass through at least one optically transparent layer 2-235 and reach the excitation region 3-215, with an emission from the sample of 1. It is possible to pass through one or more of the same layers to reach sensor 2-250.

In some embodiments, at least one surface of the sample well 2-211 is one of the materials affecting the action of the sample in the sample well, as shown in FIG. 3-8. It can be coated with multiple layers 3-211, 3-280. For example, a thin dielectric layer 3-280 (eg, alumina, titanium nitride, or silica) can be deposited as a passivation coating on the sidewalls of the sample wells. Such a coating is intended to reduce sample adhesion of the sample outside the excited region 3-215, or between the sample and the material 2-221 in which the sample wells 2-211 are formed. It can be done to reduce the interaction. The passivation coating thickness in the sample well can be between about 5 nm and about 50 nm, according to some embodiments.

In some embodiments, the material for coating layer 3-280 may be selected based on the affinity of the chemical agent for that material, layer 3-280 being treated with a chemical or biological substance. It has become possible to further prevent the adhesion of the sample species to the layer. For example, the coating layer 3-280 can consist of alumina, which can be passivated by a polyphosphonate passivation layer, according to some embodiments. Additional or alternative coating and passivating agents may be used in some embodiments.

According to some embodiments, at least the bottom surface and / or dibot 3-216 of sample well 2-211 is treated with a chemical or biological adhesive material 3-2111 (eg, biotin). , It is possible to promote sample retention. The sample can be retained permanently or temporarily, for example, for a period of time between at least about 0.5 ms and about 50 ms. In another embodiment, the adhesive material is capable of promoting temporary retention of sample 3-101 over a longer period of time. Any suitable adhesive material can be used in various embodiments and is not limited to biotin.

According to some embodiments, the layer of material 3-235 adjacent to the sample well can be selected based on the affinity of the adhesive material for the material of that layer. In some embodiments, passivation of the sidewalls of the sample wells can prevent coating of the adhesive material on the sidewalls, with the adhesive material 3-2111 being the base of the sample wells. It is designed to be preferentially deposited in. In some embodiments, the adhesive coating can extend over a portion of the sidewall of the sample well. In some embodiments, the adhesive material can be deposited by an anisotropic physical deposition process (eg, evaporation, sputtering) so that the adhesive material accumulates in the base or divot of the sample well. , And are not visibly formed on the side wall of the sample well.

Various fabrication techniques can be used to fabricate sample wells 2-211 for assay chips. Some exemplary processes are described below, but the invention is not limited to these examples.

Sample wells 2-211 can be formed by any suitable microfabrication or nanofabrication process, including, but not limited to, photolithography, deep UV photolithography, immersion photolithography, proximity field. Optical contact photolithography, EUV lithography, X-ray lithography, nanoimprint lithography, interferrometric lithography, step-and-flash lithography, direct light electron beam lithography, ion beam lithography, ion It is possible to include processing steps associated with beam milling, lift-off processing, reactive ion etching, selective photolithography, molecular self-assembly, organic synthesis and the like. According to some embodiments, the sample wells 2-211 can be formed using photolithography and lift-off processing. An exemplary fabrication process associated with sample well lift-off processing is shown in Figure 3-9. In pixels, fabrication of only a single sample well or structure is typically shown in the drawings, but multiple sample wells or structures are in parallel (eg, in each pixel) of the substrate. It will be understood that it can be made above.

According to some embodiments, the layer 3-235 (eg, an oxide layer) above the substrate is an antireflection (ARC) layer 3-910 and a photoresist, as shown in FIG. 3-9A. Can be covered by 3-920. The photoresist can be exposed and patterned using photolithography and resist development. The resist can be developed to remove exposed or unexposed parts (depending on the resist type), leaving pillars 3-922, which are shown in Figure 3-922. As shown in 9B, it has a diameter approximately equal to the desired diameter for the sample well. The height of the pillars can be greater than the desired depth of the sample wells.

The pattern of pillars 3-922 can be transmitted to ARC layer 3-910 via anisotropic reactive ion etching (RIE), for example as shown in FIG. 3-9C. The region can then be coated with at least one material 2-221, eg, a conductor or metal, desired to form a sample well. A portion of the deposited material or pieces forms a cap 3-232 above pillar 3-922, as shown in FIG. 3-9D. The resist and ARC are then subjected to a selective removal process (eg, using a chemical bath with or without agitation, at least dissolving the resist and releasing or "lifting off" the cap). It can be peeled off from the substrate. If ARC remains, it can be stripped from the substrate using selective etching, leaving sample well 3-210 as shown in FIG. 3-9E. According to some embodiments, the sidewall 3-214 of the sample well can be tilted due to the nature of the deposition of at least one material 2-221.

As used herein, "selective etching" removes or etches at a higher rate (eg, at least twice the rate) than the etchant etches other materials that are not intended to be removed. It means an etching process in which the etchant selectively etches one material that is desired to be.

Since resists and ARCs are typically polymer-based, they are considered to be soft materials, which have a high aspect ratio (eg, more than about 2: 1 with respect to height-to-width). It may not be suitable for forming sample wells with a large aspect ratio). For sample wells with higher aspect ratios, hard material may be included in the lift-off process. For example, a hard layer (eg, an inorganic material) may be deposited prior to depositing the ARC and photoresist. In some embodiments, a layer of titanium or silicon nitride may be deposited. The layer of hard material should show preferential etching above one or more materials 2-221 in which the sample wells are formed. After the photoresist is patterned, the pattern of pillars can be transmitted into the ARC and the underlying rigid material 3-930 produces a structure as shown in FIG. 3-9F. The photoresist and ARC can then be stripped, material 2-221 is deposited and a lift-off step is performed to form sample wells.

According to some embodiments, the lift-off process is to form a sample well consisting of the energy-enhanced structure 3-711, as shown in FIGS. 3-7C and 3-7D. Can be used.

An alternative process for forming sample wells is shown in Figure 3-10. In this process, the sample wells can be etched directly into at least one material 2-2111. For example, at least one material 2-211 from which a sample well will be formed can be deposited on substrate 3-325. The layer can be coated with an ARC layer 3-910 and a photoresist 3-920, as illustrated in FIG. 3-10A. The photoresist can be patterned to form holes with diameters approximately equal to the desired diameter of the sample wells, as shown in FIG. 3-10B. The hole pattern can be transmitted to the ARC through layers 3-230 using anisotropic reactive ion etching, for example as shown in FIG. 3-10C. The resist and ARC can be stripped to produce sample wells as shown in Figure 3-10D. According to some embodiments, the sidewalls of the sample well formed into the layer of material 3-230 by etching are more vertical than the sidewalls resulting from the lift-off process. Is possible.

In some embodiments, the photoresist and ARC can be used to pattern a hard mask (eg, silicon nitride or oxide layer, not shown) on material 2-221. The patterned holes can then be transmitted to the hard mask, which is then used to transfer the pattern into the layer of material 2-221. Hard masks can allow for greater etching depth into the layer of material 2-221, forming sample wells with higher aspect ratios.

The lift-off process and direct etching described above when multiple layers of different materials are used to form a stack of materials 2-211 into which the sample wells are formed. It will be recognized that fabrication techniques can be used to form sample wells. An exemplary stack is shown in Figure 2-11. According to some embodiments, the material is used to improve the coupling of the excitation energy into the excitation region of the sample well, or to reduce the transmission or re-radiation of the excitation energy into the bulk sample. Stacks can be used to form sample wells. For example, the absorption layer 3-942 may be deposited on top of the first layer 3-940. The first layer can be made of metal or metal alloy, and the absorbent layer can be made of a material that blocks surface plasmons, such as amorphous silicon, TaN, TiN, or Cr. Also, in some embodiments, the surface layer 3-944 may be deposited to passivate the surface surrounding the sample well (eg, prevent molecular attachment).

The formation of sample wells comprising Dibot 3-216 can be performed in any suitable manner. In some embodiments, the dibot may be formed adjacent to the sample well by etching and further into the adjacent layer 3-235 and / or any one or more intervening layers. For example, after forming a sample well in a layer of material 2-221, that layer 2-221 is used as an etch mask for patterning the divot, as shown in FIG. 3-12. obtain. For example, the substrate is capable of undergoing selective anisotropic reactive ion etching, allowing Dibot 3-216 to be etched into adjacent layers 3-235. For example, in embodiments where the material 2-221 is made of metal and the adjacent layer 3-235 is silicon oxide, reactive ion plasma etching with a feed gas consisting of CHF ₃ or CF ₄ is used. It is possible to preferentially remove the exposed silicon oxide below the sample well to form the dibot 3-216. As used herein, "silicon oxide" generally represents SiO _x and can include, for example, silicon dioxide.

In some embodiments, the conditions in the plasma during the etch (eg, bias and pressure on the substrate) can be controlled to determine the etch profile of the dibot 3-216. For example, at low pressures (eg, less than about 100 mTorr) and high DC bias (eg, greater than about 20V), the etching is highly anisotropic and is substantially straight, as shown in the drawings. It is possible to form a side wall of a vertical dibot. At higher pressures and lower biases, etching is more isotropic and is capable of producing tapered and / or curved divot sidewalls. In some embodiments, a wet etch can be used to form a divot, which is substantially isotropic and extends to the sidewall of the sample well, or to the sidewall of the sample well. Beyond, it is possible to form an approximately spherical divot that can extend laterally beneath material 2-221.

3-13A through 3-13C form dibots 3-216 (eg, dibots such as those shown in FIG. 3-7B) having smaller transverse dimensions than sample wells 2-211. Shows the process steps that can be used for. In some embodiments, after forming the sample wells, a conformal sacrificial layer 3-960 may be deposited on the area containing the sample wells. According to some embodiments, the sacrificial layer 3-960 can be deposited by a vapor deposition process, such as, for example, chemical vapor deposition (CVD), plasma-enhanced CVD, or atomic layer deposition (ALD). The sacrificial layer is then etched back using a first anisotropic etch selective for the sacrificial layer 3-960, removing the layer from the horizontal surface and shown in FIG. 3-13B. As such, it is possible to leave a sidewall coating 3-962 on the wall of the sample well. The etch back is selective and in some embodiments it is possible to stop on material 2-221 and adjacent layers 3-235, or in some embodiments timing is chosen. It is possible that it is a non-selective etch performed in.

A second anisotropic etch selective for the adjacent layer 3-235 is performed to etch the dibot 3-216 into the adjacent layer, as shown in FIG. 3-13C. obtain. The sacrificial sidewall coating 3-962 can then optionally be removed by selective wet or dry etch. Removal of the sidewall coating opens the sample wells to have a larger transverse dimension than Divot 3-216.

According to some embodiments, the sacrificial layer 3-960 can be made of the same material as the adjacent layer 3-235. In such an embodiment, the second etch is capable of removing at least some of the side wall coatings 3-962 when the divot is etched into the adjacent layer 3-235. .. This etch back of the sidewall coating is, in some embodiments, capable of forming the sidewall of the tapered divot.

In some embodiments, the sacrificial layer 3-960 is formed from a material used to passivate the sidewalls of the sample well (eg, reduce sample adhesion on the sidewalls of the sample well). It is possible, or it is possible to include a layer of the material. At least some of layers 3-960 can then be left on the walls of the sample wells after the formation of the dibot.

According to some embodiments, the formation of the sidewall coating 3-962 occurs after the formation of the divot. In such an embodiment, layer 3-960 coats the sidewalls of the divot. Such a process can be used to passivate the sidewalls of the divot and to localize the sample to the center of the divot.

The process steps associated with depositing the adhesive material 3-2111 on the base of sample well 2-211 and the passivation layer 3-280 are shown in FIG. 3-14. According to some embodiments, the sample well can include a first passivation layer 3-280 over the wall of the sample well. The first passivation layer can be formed, for example, as described above in connection with FIG. 3-13B or FIG. 3-8. In some embodiments, the first passivation layer 3-280 can be formed by any suitable deposition process and etch back. In some embodiments, the first passivation layer can be formed by oxidizing the material 3-230 into which the sample wells are formed. For example, the sample wells can be formed from aluminum, which can be oxidized to form a coating of alumina on the sidewalls of the sample wells.

The adhesive substance 3-980 or the adhesive substance precursor (eg, the material that preferentially binds to the adhesive substance) is an anisotropic physical deposition process, as shown in FIG. 3-14A. For example, vapor deposition can be used to deposit on a substrate. The adhesive material or the adhesive material precursor can form an adhesive material layer 3-2111 at the base of the sample well, as shown in FIG. 3-14B, which is the sample well. It is possible to coat the upper surface of the material 2-221 formed therein. Subsequent angled directional deposits (sometimes referred to as shadow deposits or shadow evaporation processes), shown in FIG. 3-14C, do not cover the adhesive layer 3-211. , Can be used to deposit a second passivation layer 3-280 on the upper surface of material 2-221. During the shadow deposition process, the substrate can be rotated around an axis perpendicular to the substrate, and the second passivation layer 2-280 deposits more evenly around the upper rim of the sample well. It has become like. The resulting structure is shown in Figure 3-14D, according to some embodiments. As an alternative to depositing a second passivation layer, a flattening etch (eg, a CMP step) can be used to remove the adhesive material from the upper surface of material 2-221.

According to some embodiments, the adhesive layer 3-211 can be centrally deposited at the base of the tapered sample well, as shown in FIG. 3-15. For example, the adhesive or adhesive precursor is contained in a tapered sample well formed as described above, as shown in FIG. 3-14A. Can be deposited in one direction. The walls of the sample wells can be passivated by an oxidation process before or after the deposition of adhesive layer 3-2111. Adhesives or precursors remaining on the surface of material 2-221 can be passivated as described in connection with FIG. 3-14D. In some embodiments, the adhesive material on the upper surface of material 2-221 can be removed by a chemical and mechanical polishing step. By centrally forming an adhesive layer or adhesive layer precursor at the base of the sample well, a detrimental effect on emissions from the sample (eg, suppression or quenching of sample radiation from the sample wall, sample). Unfavorable radiation distribution from the sample due to the sample not being centered to the energy coupling structure formed around the well, adverse effects on the emission lifetime of the sample) can be reduced. ..

In some embodiments, the lift-off patterning, etching, and deposition processes used to form sample wells and divots are used to form an integrated CMOS circuit on a sensor chip. It is possible that it is compatible with the CMOS process. Thus, sensors can be manufactured using conventional CMOS equipment and fabrication techniques, but in some embodiments custom or specialized fabrication equipment can also be used.

Modifications of the process steps described above can be used to form alternative embodiments of sample wells. Tapered sample wells, such as those shown in Figure 3-7A or Figure 3-7B, use the angled deposition process shown in Figure 3-14C. Can be formed. For the sample wells in Figure 3-7B, the angle of deposition can be varied during the deposition process. For such embodiments, sample wells with substantially straight and vertical sidewalls are first formed, then additional material 2-221 is deposited by angled deposition and sampled. -It is possible to taper the side wall of the well.

B. Coupling of Excitation Energy to Sample Wells As shown in Figures 2-1, 2-3, the excitation energy 2-251 from the excitation source 2-250 is the component of device 2-120 and the assay chip 2-110. Guided to sample wells 2-211 using the components of. This section describes the components of assay chip 2-110 that can facilitate the coupling of excitation energies 2-251 to sample wells 2-211.

Energy coupling from the excitation source to the sample well is improved or affected by forming an excitation coupling structure within and / or adjacent to the sample well. Can be affected. Excited coupling structures can consist of micro-scale or nano-scale structures made around sample wells in some embodiments, or in some embodiments. , Can consist of structures or particles formed in the sample wells. The excitation coupling structure can affect the radiative excitation of the sample in some embodiments and can affect the non-radiative excitation of the sample in some embodiments. In various embodiments, the radiated excitation coupling structure is capable of increasing the intensity of the excitation energy within the excitation region of the sample well. The non-radiatively excited coupling structure is capable of improving and / or altering the non-radiant energy transfer path from the excited source (which can be radiant or non-radiative) to the sample.

C. Radiation Excited Coupling Structures There are several different types of radiation excited coupling structures to affect the coupling of excitation energy from the excitation source to the excitation region in the sample well. Can be used. Some radiated coupling structures can be formed from conductors (eg, including metal layers) and also locally affect the excitation energy near and / or within the sample wells. Supports surface plasmon vibration (which locally modifies the electromagnetic field). In some cases, the surface plasmon structure is capable of increasing the excitation energy in the excitation region of the sample well more than twice. Some radiative coupling structures are capable of altering the phase and / or amplitude of the excitation field to enhance the excitation energy in the sample wells. Various embodiments of the radiated excitation coupling structure are described in this chapter.

FIG. 4-1A shows just one example of a surface plasmon structure 4-120 that can be used to enhance the coupling of excitation energy into the sample wells. The drawings show a plan view of the region around the surface plasmon structure 4-120 and represent the results of a numerical simulation of the electric field strength around the structure. The drawing shows a surface plasmon structure consisting of three triangular features with sharp vertices located very close to the sample well (not shown). According to some embodiments, the surface plasmon structure is patterned with a metal or conductor (eg, any one of the following metals or metal alloys: Al, Au, Ag, Ti, TiN, or a combination thereof: It is possible to consist of a thin film). The thickness of the membrane can be between about 10 nm and about 100 nm in some embodiments, but in other embodiments other thicknesses may be used. In some embodiments, the surface plasmon structure is capable of containing sharp features 4-110 that are located very close to the sample well (eg, within about 100 nm).

FIG. 4-1B shows a cross-sectional elevation view of the surface plasmon structure of FIG. 4-1A taken along the dotted line. The simulation shows a localized high intensity region 4-505 of the excitation energy adjacent to the vertices of the triangle of the surface plasmon structure. For this simulation, the surface plasmon structure 4-120 was positioned on the dielectric layer 4-135 (silicon dioxide). The surface plasmon structure taps the energy from the evanescent field of the waveguide to increase the strength in the sample well.

In some embodiments, the enhancement of excitation energy by the surface plasmon structure can be localized to the extent that deep sample wells 2-211 are not required. For example, deep sample wells are not required if the high intensity region 4-505 is formed to have a diameter of approximately 100 nm and has a peak intensity value greater than about 80% of the intensity outside the region. there is a possibility. Only samples within the high intensity region 4-505 will contribute significant emissions for detection purposes.

When the incident electromagnetic field interacts with the surface plasmon structure, a surface wave current is generated in the structure. The shape of the structure can affect the strength and distribution of these surface plasmons. These localized currents interact with and significantly alter the incident electromagnetic field in the immediate vicinity of the surface plasmon structure, for example, as shown by the high intensity region 4-505 in FIG. 4-1B. And it is possible to increase it. In some embodiments, an emitter that emits energy near a surface plasmon structure (eg, a fluorescent tag) can have its emissions modified by the structure, and far-field radiation from the emitter. The pattern is changed.

Another embodiment of the surface plasmon structure 4-122 is shown in the plan view of FIG. 4-1C. The bow tie structure shown can consist of two triangular metal structures located adjacent to sample wells 2-211. The structure can be patterned, for example, below the sample well and / or adjacent to the excited region of the sample well. In some embodiments, it is possible that a gap 4-127 is present between the sample well and the sharp feature 4-125 of the surface plasmon structure. The gap 4-127 can be between approximately 10 nm and approximately 200 nm, according to some embodiments. In some embodiments, the gap 4-127 can be between approximately 10 nm and approximately 100 nm. Sharp features 4-125 can consist of points or sharp bends within the edges of the surface plasmon structure, as shown in the drawings. Sharp features can have any suitable shape. In some embodiments, the bend radius of the sharp feature 4-125 can be less than approximately five wavelengths associated with incident excitation energies. In some embodiments, the bend radius of the sharp feature 4-125 can be less than approximately two wavelengths associated with the incident excitation energy. In some embodiments, the bend radius of the sharp feature 4-125 can be less than approximately five wavelengths associated with surface plasmon waves excited by incident excitation energies. In some embodiments, the bend radius of the sharp feature 4-125 can be less than approximately two wavelengths associated with a surface plasmon wave excited by incident excitation energies.

According to some embodiments, the surface plasmon structure 4-122 can be patterned in sample wells 2-211, as illustrated in the elevation of FIG. 4-1D. In some embodiments, the surface plasmon structure within the sample well is patterned with one or more fingers (eg, for example) on the sidewall of the sample well, as shown in the drawing. It can consist of metal fingers). FIG. 4-1E shows a plan view of the sample well 2-211 showing the surface plasmon structure 5-122 formed on the side wall portion in the sample well. In some embodiments, the lower ends of these surface plasmon structures 4-122 form sharp features or bends, where the electromagnetic fields are enhanced. The surface plasmon structure 4-122 may or may not extend to the base of the sample well.

In some embodiments, the surface plasmon structure 4-122 may be arranged to affect the polarization of the excitation energy and / or the energy emitted from the sample well. For example, a pattern as shown in FIG. 4-1E is a preferred orientation of linear or elliptical excitation polarization and / or linear or elliptical polarization from an emitter in a sample well. It can be used to influence the preferred orientation.

The surface plasmon structure can be patterned in a shape other than that shown in FIGS. 4-1A to 4-1E. For example, the surface plasmon structure can be patterned as a regular or periodic structure, as shown in FIG. 4-2A, according to some embodiments. For example, the surface plasmon structure can be patterned as an array of protruding features 4-210 on the underside surface of material 2-221 in which sample wells 2-211 are formed. Periodic surface plasmon structures can be formed in regular arrays such as gratings, grids, grids, circular gratings, spiral gratings, oval gratings, or any other suitable structure. .. It is possible that there is a substantially uniform spacing between the protrusions 4-210 of the surface plasmon structure. In some embodiments, the spacing can have any value between approximately 40 nm and approximately 250 nm. According to some embodiments, the protrusion can have a height h between about 20 nm and about 100 nm. In some embodiments, the spacing s may be non-uniform or chirped (has a decreasing value over a larger radial distance). In some embodiments, the protrusions 5-210 of the surface plasmon structure can be patterned as Fresnel zone plates. According to some embodiments, the surface plasmon structure of 4-210 can be formed adjacent to a transparent layer and / or a dielectric layer 3-235. In some embodiments, the gap (spacing) between the protrusions 4-210 may be periodic, and in other embodiments the protrusions 4-210 may be aperiodic.

In some embodiments, the surface plasmon structure 4-212 is spaced from the material 2-221 in which the sample wells are formed, as shown in FIG. 4-2B. obtain. For example, it is possible that an intervening dielectric layer 4-247 exists between the surface plasmon structure 4-212 and the material 4-230. According to some embodiments, the surface plasmon structure 4-212 may be positioned adjacent to the sample well dibot 3-216, as shown in the drawings. For example, the surface plasmon structure 4-212 may be positioned adjacent to the sidewall of the Divot 3-216, as shown in FIG. 4-2B.

FIG. 4-2C illustrates the surface plasmon structure 4-214 formed as concentric circular gratings. Structure 4-214 can consist of concentric conductive rings 4-215, according to some embodiments. The rings can be separated by regular spacing s and can have a height h as described in connection with FIG. 4-2A. According to some embodiments, a sample well 4-210 with a voluntary dibot can be located in the center of the ring. The circular grating can be patterned adjacent to the base of the sample well.

The periodicity of the surface plasmon structure can be selected to form a resonant structure, according to some embodiments. For example, the spacing s of the surface plasmon structure may be selected to be approximately half the wavelength of the surface plasmon wave generated in the structure by the excitation energy. When formed as a resonance structure, the surface plasmon structure is capable of accumulating and resonating excitation energy along the direction of the periodic surface plasmon structure. Such resonance behavior can increase electromagnetic energy in or adjacent to the sample well, as shown in FIG. 4-2D. The spacing of the surface plasmon structure may be periodic in some embodiments, but may be aperiodic in other embodiments. The use of aperiodic spacing allows the increase in electromagnetic fields to be specially designed for the wavelength of excitation energy and the associated wavelength of emission energy. FIG. 4-2D shows numerically simulated electromagnetic field results at the base of the sample well and around the periodic surface plasmon structure. The surface plasmon structure 4-216 is positioned adjacent to the material 2-221 in which the sample well is formed and is adjacent to the base of the sample well 2-211. The surface plasmon structure can be in the form of repeating or circular gratings at regular or irregular spacing intervals in areas away from the sample wells and outside the simulated areas. Is. For example, it is possible that there is a grating overhang of the surface plasmon structure 4-216 that is repeated between 3 and 50 times. High intensity regions 4-240 can be seen at the base of sample wells 2-211. The strength within this region can be increased more than double on the surrounding region just below the surface plasmon structure.

FIG. 4-2E shows in elevation an alternative embodiment of the resonant surface plasmon structure 4-218. According to some embodiments, the surface plasmon structure can be formed as a periodic or aperiodic grating or grid pattern and can be patterned in multiple layers 4-247. Sample wells 2-211, according to some embodiments, can be patterned through multiple layers 4-247 and within the resonant surface plasmon structure 4-218. In some embodiments, the resonant surface plasmon structure can consist of a separate conductive element 4-222, as shown in the plan view of FIG. 4-2F. In some embodiments, the resonant surface plasmon structure can consist of a continuous lattice pattern 4-250, as shown in FIG. 5-2G. The dielectric filler 4-252 can be positioned within the voids of the conductive material 4-250 and the sample wells 2-211 can be positioned with the voids.

There are a variety of different surface plasmon structures that can be used to enhance coupling into the sample wells or to influence emissions from the samples in the sample wells. FIG. 4-2H shows a further alternative embodiment of the surface plasmon structure in plan view. An elevation view of the structure is shown in FIG. 4-2I. According to some embodiments, the surface plasmon structure can consist of an array of disks dispersed around sample wells 2-211. In some embodiments, instead of using a conductive disk 4-260, the surface plasmon structure can consist of a conductive layer, and a pattern of dispersed holes is formed through the conductive layer. Such a structure may be referred to as a "nano antenna".

A variety of different processes can be used to pattern surface plasmon structures adjacent to sample wells. 4-3A through 4-5E show structures associated with process steps that can be used to form surface plasmon structures adjacent to sample wells, according to some embodiments. .. Referring here to FIG. 4-3A, the process for forming a surface plasmon structure forms a resist layer 4-310 on an antireflection coating (ARC) 4-320 on a masking layer 4-330. It is possible to consist of things. The layer may be disposed on top of the transparent dielectric layer 3-235, according to some embodiments. The resist layer 4-310 can consist of a photoresist or electron-beam resist or ion-beam resist that can be patterned by lithography. The masking layer 4-330 can consist of a hard mask made of an inorganic material (eg, silicon or silica nitride, or any other suitable material), according to some embodiments. be.

In some embodiments, a photolithographic process can be used to pattern resist 4-310, as shown in FIG. 4-3B. The pattern selected can consist of a layout of protrusions or holes that will be used to form the desired surface plasmon structure. After development of resist 4-310, the area of ARC will be exposed and the pattern can be etched into ARC layer 4-320 and then into masking layer 4-330. The resist and ARC can be stripped from the substrate and the resulting structure can appear as shown in FIG. 4-3C. The masking layer 4-330 can then be used as an etch mask and the pattern is the underlying dielectric layer 3 via selective anisotropic etching, as shown in FIG. 4-3D. It can be transmitted into -235.

A layer of conductive material 2-221, or a material consisting of a conductor, can then be deposited over the region, as illustrated in FIG. 4-3E. Any suitable conductive material can be used to form a surface plasmon structure, whether or not it is deposited as a separate layer from material 2-221. For example, in some cases, the first conductive material may be deposited as the base layer of material 2-221 in which the surface plasmon structure is formed. Examples of materials that can be used to form surface plasmon structures include, but are not limited to, Au, Al, Ti, TiN, Ag, Cu, and alloys, or a combination layer thereof.

Material 2-221, or a layer of material, can be deposited by any suitable deposition process, including but not limited to physical deposition processes or chemical vapor deposition processes. In some embodiments, the material 2-221 is capable of having a thickness between approximately 80 nm and approximately 300 nm. In some embodiments, material 2-221 can be flattened (eg, using a CMP process), but flattening is not required. Sample wells can be formed in material 2-221 using any suitable process described herein in connection with making sample wells.

We have found that forming a surface plasmon structure according to the steps shown in FIGS. 4-3A to 4-3E may require precise alignment of the sample wells to the surface plasmon structure. I realized that there is. For example, as shown in FIG. 4-2C, a surface plasmon structure consisting of concentric gratings requires accurate alignment of sample wells 2-211 with respect to the center of the surface plasmon structure 4-214. there is a possibility. To avoid the manufacturing difficulties associated with such precise alignment, the self-alignment process shown in FIGS. 4-4A-4E may be used.

Referring here to FIG. 4-4A, the process for forming the surface plasmon structure and the sample well self-aligned with the surface plasmon structure is a masking layer on top of the transparent dielectric layer 2-235. It can consist of forming 4-410. The masking layer can consist of a hard mask made of an inorganic material, such as silicon or silica nitride, according to some embodiments. The thickness of the masking layer 4-410 can be approximately equal to the desired height of the sample well 2-212. For example, the thickness of the masking layer can be between approximately 50 nm and approximately 200 nm according to some embodiments, but in other embodiments other thicknesses may be used.

The masking layer 4-410 can be patterned to generate a void 4-430 having the desired pattern of surface plasmon structure to be patterned in the dielectric layer 2-235. The patterning of the masking layer 4-410 can be performed by any suitable lithography process (eg, photolithography, electron-beam lithography, ion-beam lithography, EUV lithography, X-ray lithography). The resulting structure can appear as shown in Figure 4-4B. The structure can include central pillars 4-420, which will then be used to form self-aligned sample wells.

The resist 4-440 (eg, photoresist) can then be patterned onto the patterned masking layer 4-410, as shown in FIG. 4-4C. The alignment for patterning resist 4-440 (eg, the alignment of the mask with respect to the substrate) does not have to be highly accurate, just to cover the central pillar 4-420 and to the surface plasmon structure. Resist 4-440 is required to not coat the void 4-430 that will be used to form.

Selective anisotropic etching is then performed to etch the dielectric layer 2-235 and in the dielectric, as shown in FIG. 4-4D, according to some embodiments. It can be used to convey the pattern of surface plasmon structures to. A selective isotropic etch can then be used to remove the exposed portion of the masking layer 4-410. The isotropic etch can be, for example, a wet etch, but in some embodiments, an isotropic dry etch can also be used. Since the resist 4-440 covers the central pillar 4-420, the central pillar will not be etched and will remain on the substrate, as shown in FIG. 4-4E. The resist 4-440 can then be stripped from the substrate to expose pillars 4-420, as shown in FIG. 4-4F.

Then, according to some embodiments, a metal conductive material 2-221, or a stack of materials containing the conductive material, is then deposited onto the region as illustrated in FIG. 4-4G. obtain. The central pillar 4-420 and the cap of material deposited on the pillar are then removed by selective wet etching of the pillar and the cap can be lifted off. Removal of the central pillar leaves sample wells self-aligned with the underlying surface plasmon structure 4-450.

An alternative process can be used to form a sample well that is self-aligned with the surface plasmon structure, which is shown in FIGS. 4-5A-4-5E. According to some embodiments, one or more conductive layers 4-510, 4-520 are transparent using any suitable lithographic process, as shown in FIG. 4-5A. Can be patterned on the dielectric layer 2-235. In some embodiments, the first layer 4-510 can be made of aluminum and the second layer 4-520 can be made of titanium nitride, but in various embodiments. , Other material combinations may also be used. The total thickness of one or more layers can be approximately equal to the desired height of the sample wells, according to some embodiments. Patterning can form sample wells 2-211 and voids 4-525 adjacent to sample wells in one or more metal layers. Voids can be placed within the pattern of the desired surface plasmon structure.

In some embodiments, the dielectric layer 2-235 is such that the pattern of the surface plasmon structure and sample well 2-211 is transmitted into the dielectric layer, as shown in FIG. 4-5B. Can be etched into. The etch depth into the dielectric can be between approximately 20 nm and approximately 150 nm, according to some embodiments. Resist 4-440 can be patterned to cover the sample wells, as shown in FIG. 4-5C. The alignment for patterning the resist does not have to be highly accurate and covers the etched adjacent regions of the dielectric layer 2-235 that will be used to form the surface plasmon structure. No need to cover the sample wells.

As illustrated in FIG. 4-5D, a layer of conductive material 4-512, or material containing conductors, can be deposited onto the region using any suitable deposition process. Material 4-512 can fill the etched areas of the dielectric layer and can extend over one or more layers 4-510, 4-520. The resist 4-440 and the material covering the resist can then be removed according to a lift-off process. The resulting structure, shown in FIG. 4-5E, leaves sample wells self-aligned with the surrounding surface plasmon structure. Sample wells include Divot 3-216.

In some embodiments, the process shown in FIGS. 4-5A-4-5E can be used to form sample wells without Divot 3-216. For example, resist 4-440 may be patterned onto sample wells 2-211 before the dielectric layer 2-235 is etched. The dielectric layer 2-235 can then be etched, which will transfer the pattern of the surface plasmon structure to the dielectric layer, but will not form a dibot. The process can then proceed as illustrated in FIGS. 4-5D and 4-5E to produce self-aligned sample wells without divot.

In addition to or as an alternative to the surface plasmon structure, other structures can be patterned near sample wells 2-211 to increase the excitation energy in the sample wells. Is. For example, some structures are capable of altering the phase and / or amplitude of the incident excitation field, increasing the intensity of the excitation energy in the sample wells. FIG. 4-6A shows a thin lossy film 4-610, in which the thin lossy film 5-610 is used to change the phase and amplitude of the incident excitation energy, as well as the electromagnetic radiation in the sample wells. Can be used to increase the strength of.

According to some embodiments, the thin lossy membrane can generate intensifying interference of excitation energies, resulting in field enhancement in the excitation region of the sample well. FIG. 4-6B shows a numerical simulation of the excitation energy incident on the sample well, where a thin lossy film 4-610 is formed directly adjacent to the sample well. For simulation, the sample wells have a diameter of approximately 80 nm and are formed in a metal layer of gold approximately 200 nm thick. The sample well consists of SCN and suppresses the propagation of excitation energy through the sample well. The thin lossy film 4-610 is approximately 10 nm thick, is made of germanium, and covers the underlying transparent dielectric made of silicon dioxide. A thin lossy membrane extends across the entrance aperture of the sample well. Simulations show that the intensity of the excitation energy is the highest at the entrance aperture of the sample well. The intensity of the excitation energy in this bright region 4-620 is greater than twice the intensity value for the left and right of the sample well.

The thin lossy membrane can be made from any suitable material. For example, a thin lossy film can be made from a material having a refractive index n approximately the same as the extinction coefficient k for the material. In some embodiments, the thin loss film can be made from a material whose index of refraction n is within about two orders of magnitude from the value of the material's extinction coefficient k. Non-limiting examples of such materials at visible wavelengths are germanium and silicon.

The thin lossy membrane can be of any suitable thickness, which can depend on one or more characteristic wavelengths associated with one or more excitation sources. In some embodiments, the thin lossy membrane can be between approximately 1 nm and approximately 45 nm in thickness. In other embodiments, the thin lossy membrane can be between approximately 15 nm and approximately 45 nm in thickness. In yet another embodiment, the thin lossy membrane can be between approximately 1 nm and approximately 20 nm in thickness.

The effect of the thin loss film on the reflectance from the material 2-221 in which the sample well is formed, the excitation energy loss in the thin loss film, and the excitation energy loss in the material 2-221. Is shown in the graph of FIG. 4-6C. One curve plotted in the graph represents the reflectance curve 4-634, and material 2-221 and thin loss as the thickness of the thin loss film changes from 0 nm to 100 nm. It shows how the reflectance from film 4-610 changes. The reflectance reaches a minimum at about 25 nm, according to the simulated embodiment. The minimum reflectance will occur at different thicknesses depending on the characteristic wavelength of the excitation energy and the material used for the thin lossy film and material 2-221. In some embodiments, the thickness of the thin lossy membrane is selected so that the reflectance is approximately its minimum.

In some embodiments, the thin loss film 4-610 may be spaced from sample wells 2-211 and material 2-221 as shown in FIG. 4-6D. For example, a thin dielectric layer 4-620 (eg, silicon oxide SiO _x ) can be formed on a thin lossy film, and sample wells 2-211 are adjacent to the dielectric layer 4-620. Can be formed. The thickness of the dielectric layer 4-620 can be between about 10 nm and about 150 nm according to some embodiments, but in some embodiments other thicknesses may be used. ..

Although shown as a single layer, a thin lossy membrane can consist of multiple layers of two or more materials. In some embodiments, a multi-layer stack consisting of alternating layers of thin lossy film 4-610 and dielectric layer 4-620 is flanked by sample wells 2-211, as shown in FIG. 4-6E. Can be formed. The thickness of the thin loss film 4-610 in the stack of layers can be between approximately 5 nm and approximately 100 nm, and the thickness of the dielectric layer 4-620 in the stack is several. According to the embodiment, it is possible to be between about 5 nm and about 100 nm. In some embodiments, the multi-layer stack may consist of a layer of silicon dioxide (thickness 4.3 nm), a layer of silicon (thickness 14.35 nm), and a layer of germanium (thickness 6.46 nm). Although possible, in other embodiments, other thicknesses may be used. In some embodiments, the multi-layer stack comprises a layer of silicon dioxide (approximately 4.2 nm thick), a layer of silicon (approximately 14.4 nm thick), and a layer of germanium (approximately 6.5 nm thick). Although it is possible to consist of, in other embodiments, other thicknesses may be used.

Thin lossy membranes can be made from any suitable material that exhibits at least some loss to incident radiation. In some embodiments, the thin loss film can consist of semiconductor materials such as silicon and germanium, but other materials can also be used. In some embodiments, the thin lossy membrane can consist of an inorganic material or metal. In some embodiments, the thin lossy membrane can consist of an alloy or compound semiconductor. For example, a thin lossy membrane can be made of an alloy containing Si (57.4% by weight), Ge (25.8% by weight), and SiO ₂ (16.8% by weight), but other In embodiments, other ratios and compositions may also be used.

According to some embodiments, the thin lossy membrane uses any suitable blanket deposition process, such as, for example, a physical deposition process, a chemical vapor deposition process, a spin-on process, or a combination thereof. Can be formed on a substrate. In some embodiments, the thin lossy membrane can be treated after deposition, for example, burned, annealed, and / or subject to ion implantation.

Other phase / amplitude changing structures can be used in addition or alternatives to enhance the excitation energy in the sample wells. According to some embodiments, and as shown in FIG. 4-7A, the reflection stack 4-705 may be spaced from the sample wells 2-2111. In some embodiments, the reflective stack can consist of a dielectric stack of materials with alternating refractive indexes. For example, the first dielectric layer 4-710 can have a first refractive index, and the second dielectric layer 4-720 has a second refractive index different from the first refractive index. It is possible to have. The reflection stack 4-705 is capable of exhibiting high reflectivity with respect to excitation energy and low reflectivity with respect to radiated emissions from emitters in sample wells in some embodiments. Is. For example, the reflection stack 4-705 can exhibit greater than approximately 80% reflectivity with respect to excitation energy and can exhibit less than approximately 40% reflectivity with respect to emissions from the sample. However, in some embodiments, other reflectivity values may also be used. The dielectric layer 4-730 that transmits the excitation energy can be positioned between the reflection stack and the sample well.

According to some embodiments, the reflection stack 4-705 shown in FIG. 4-7A forms a resonator with the material 2-221 in which the sample wells 2-211 are formed. It is possible. For example, the reflection stack may be spaced from material 2-221 by a distance approximately equal to half or an integral multiple of the wavelength of the excitation energy in the dielectric material 4-730. By forming the resonator, the excitation energy can pass through the reflection stack, resonate, and increase in the space between the material 2-221 and the reflection stack 4-705. It is possible to increase the excitation intensity in sample wells 2-211. For example, the intensity can be increased in the resonant structure by a factor of 2 or more in some embodiments, a factor of 5 or more in some embodiments, and a factor of 10 or more in some embodiments. ..

Additional structures may be added near the sample wells, as shown in FIGS. 4-7B and 4-7C. According to some embodiments, the dielectric plug 4-740 having a first index of refraction higher than the second index of refraction of the dielectric layer 4-730 is as shown in FIG. 4-7B. , Can be formed adjacent to sample wells 2-211. The plug can be in the shape of a cylinder with a diameter approximately equal to the diameter of the sample well, but other shapes and sizes can also be used. Due to its higher index of refraction, the dielectric plug 4-740 is capable of concentrating the excitation radiation and guiding the excitation energy towards the sample wells.

Dielectric structures such as plug 4-740 can be used with or without reflective stack 4-705, according to some embodiments. Such a dielectric structure may be referred to as a dielectric resonance antenna. The dielectric resonance antenna can have any suitable shape, for example, cylindrical, rectangular, square, polygonal, trapezoidal, or pyramidal.

4-7C and 4-7D show a photonic band gap (PBG) structure that can be formed in the vicinity of sample wells 2-211, according to some embodiments. The photonic bandgap structure can consist of a regular array or grid of optical contrast structures 4-750. The optical contrast structure can consist of a dielectric material having a refractive index different from that of the surrounding dielectric material, according to some embodiments. In some embodiments, the optical contrast structure 4-750 can have a different loss value than the surrounding medium. In some embodiments, sample wells 2-211 can be positioned as defects in the grid as shown in FIG. 4-7D. According to various embodiments, defects in the photonic lattice are capable of confining photons within the region of the defect, which can enhance the intensity of the excitation energy in the sample wells. .. The confinement resulting from the photonic bandgap structure can be substantially two-dimensional in the transverse direction of the surface of the substrate. When combined with the reflection stack 4-705, the confinement can be three-dimensional in the sample well. In some embodiments, the photonic bandgap structure can be used without a reflective stack.

Various methods have been considered for making the excited coupling structures shown in FIGS. 4-6A through 4-7D. Structures that require thin planar films (eg, dielectric films with alternating refractive indexes) can be formed by a planar deposition process, according to some embodiments. The planar deposition process can consist of physical deposition (eg, electron beam evaporation or sputtering) or chemical vapor deposition process. Separately formed in a three-dimensional shape, such as the dielectric resonance antenna 4-740 shown in FIG. 4-7B, or the optical contrast structure 4-750 shown in FIG. 4-7C. Structures that require an embedded dielectric use a lithography patterning and etching process to etch the pattern into the substrate, and, for example, subsequent deposition of the dielectric layer, and , Can be formed using substrate flattening. Also conceivable is a self-alignment processing technique for forming a dielectric resonance antenna and a photonic bandgap structure near sample wells 2-211.

4-8A through 4-8G are just one self that can be used to form a photonic bandgap structure and self-aligned sample wells as illustrated in FIG. 4-7C. It shows the structure associated with the process process for the alignment process. According to some embodiments, the reflective stack 4-705 can first be formed on a substrate on the dielectric layer 3-235, as illustrated in FIG. 4-8A. A second dielectric layer 4-730 can then be deposited on the reflective stack. The thickness of the dielectric layer 4-730 can be approximately equal to about half the wavelength of the excitation energy in the material or an integral multiple thereof. The process steps described in connection with FIGS. 4-4A to 44E were then etched with respect to the pillars 4-420 on the dielectric layer 4-730 and the photonic bandgap structure. It can be performed to form the pattern of feature 4-810. The etched features can extend into the dielectric layer 4-730 and optionally into the reflective stack 4-705. The resulting structure can appear as shown in FIG. 4-8A.

The resist 4-440 covering the pillars 4-420 can be stripped from the substrate and conformal deposition is performed as shown in FIG. 4-8B, filling the material 4- with etched features. It can be filled by 820. Filling material 4-820 can be the same material used to form pillars 4-420, according to some embodiments. For example, the filling material 4-820 and pillar 4-420 can be formed from silicon nitride, and the dielectric layer 4-730 can be made of an oxide, for example SiO ₂ .

Anisotropic etching can then be performed to etch back the packing material 4-820. The filler may be etched back, according to some embodiments, to expose the surface of the dielectric layer 4-730, resulting in a structure as shown in FIG. 4-8C. It is possible. The etch can leave pillars 4-830, which consist of the original pillars 4-420 and the sidewalls 4-822 remaining from the filling material 4-820.

Resist 4-440 can then be patterned onto the substrate as shown in FIG. 4-8D. For example, the resist can be coated on the substrate, holes can be patterned in the resist, and the resist can be developed to cut open areas in the resist around pillars 4-830. The alignment of the holes with respect to the pillars does not have to be highly accurate and the pillars 4-830 do not expose the underlying photonic bandgap structure embedded within the dielectric layer 4-730. It only needs to be exposed.

After the pillars 4-830 are exposed, an isotropic etch can be used to reduce the transverse dimensions of the pillars. According to some embodiments, the resulting pillar shape can appear as shown in FIG. 4-8E. The resist 4-440 can then be stripped from the substrate and material 2-221, or a layer of material, can be deposited onto the region. In some embodiments, material 2-221 is etched back using a CMP process and is capable of flattening the region as shown in FIG. 4-8F. A selective dry or wet etch may then be used to remove the remaining pillar structures, leaving sample wells 2-211, as illustrated in FIG. 4-8G. It is possible. As shown in the drawings, the sample wells 2-211 are self-aligned with the photonic bandgap structure patterned in the dielectric layer 4-730.

As an alternative process, the filling material 4-820 can consist of a different material than the material used to form pillars 4-420. In this process, the steps associated with FIGS. 4-8D and 4-8E may be omitted. As shown in FIG. 4-8F, after deposition and flattening of material 2-221, selective etching is performed to remove pillars 4-420. It is possible to leave the sidewalls of the filling material 4-820 lining the sample wells 2-211.

D. Non-radiatively Excited Coupling Structure The present invention provides a non-radiatively excited coupling structure for excitation energy with a sample in a sample well. Only one embodiment of the non-radiative coupling structure is shown in FIG. 4-9A. According to some embodiments, the non-radiative coupling structure can consist of semiconductor layers 4-910 formed directly adjacent to sample wells 2-211. The semiconductor layer 4-910 can be an organic semiconductor in some embodiments, or can be an inorganic semiconductor in some embodiments. In some embodiments, the dibot 3-216 may or may not be formed in the semiconductor layer. The semiconductor layer 4-910 can have a thickness between about 5 nm and about 100 nm according to some embodiments, but in some embodiments other thicknesses can be used. According to some embodiments, the excitation energy or photon 4-930 from the excitation source can collide with the semiconductor layer 4-910 to create Exiton 4-920. Exciton can diffuse to the surface of the sample well, where they can recombine non-radiatively and transfer energy to the sample adjacent to the wall of the sample well.

FIG. 4-9B shows another embodiment in which the semiconductor layer 4-912 is used and it is possible to transfer energy from the excitation energy to the sample non-radiatively. In some embodiments, the semiconductor layer 4-912 can be formed at the bottom of the sample wells or in the divot of the sample wells 2-211, as shown in the drawings. The semiconductor layer 4-912, according to some embodiments, is a directional deposition process as described herein in connection with a process step for depositing an adhesive material at the base of a sample well. Can be formed in the sample wells by using. The semiconductor layer 4-912 can have a thickness between about 5 nm and about 100 nm, according to some embodiments, but in other embodiments, other thicknesses may be used. Incident radiation can generate excitons in the semiconductor layer, which can then diffuse to the bottom surface of sample wells 2-2111. Exciton can then transfer energy non-radiatively to the sample in the sample well.

The invention also provides multiple non-radiative paths for transferring excitation energy to the sample. According to some embodiments, and as shown in FIG. 4-9C, energy transfer particles 4-940 can be deposited in the sample wells. The energy transfer particles can consist of quantum dots in some embodiments, or molecules in some embodiments. In some embodiments, the energy transfer particles 4-940 can be functionalized on the surface of the sample well through linking molecules. A thin semiconductor layer 4-910 can be formed adjacent to or in the sample well, and the exciton is a semiconductor layer from the excitation energy incident on the semiconductor layer, as shown in the drawings. Can be generated in. Exciton can diffuse to the surface of the sample well and transfer energy non-radiatively to the energy transfer particles 4-940. The energy transfer particles 4-940 can then non-radiatively transfer energy to sample 3-101 in the sample well.

According to some embodiments, it is possible that more than one energy transfer particle 4-940 is present in the sample well. For example, a layer of energy transfer particles 4-942 can be deposited in a sample well, such as the sample well shown in FIG. 4-9C.

In some embodiments, the energy transfer particles can absorb the incident excitation energy and then re-emit the radiant energy at a wavelength different from the wavelength of the absorbed excitation energy. The re-emitted energy can then be used to excite the sample in the sample well. FIG. 4-9E represents a spectral graph associated with down-converting energy transfer particles. According to some embodiments, the energy transfer particles to be down-converted consist of quantum dots, which absorb short wavelength radiation (higher energy) and one or more longer wavelength radiation (more). It is possible to release low energy). An exemplary absorption curve 4-952 is shown in the graph as a dotted line for quantum dots with radii between 6 nm and 7 nm. The quantum dots are the first band of radiation illustrated by curve 4-954, the second band of radiation illustrated by curve 4-956, and the second band of radiation illustrated by curve 4.958. It is possible to emit 3 bands.

In some embodiments, the energy transfer particles are capable of up-converting energy from the excitation source. FIG. 4-9F shows the spectrum associated with the up-conversion from the energy transfer particles. According to some embodiments, the quantum dots can be excited by radiation at approximately 980 nm and then re-emitted into one of the three spectral bands as illustrated in the graph. It is possible. The first band can be centered at approximately 483 nm, the second band can be centered at approximately 538 nm, and the third band can be centered at approximately 642 nm. The photons re-emitted from the quantum dots are of higher energy than the photons of radiation used to excite the quantum dots. Therefore, the energy from the excitation source is up-converted. One or more of the emitted spectral bands can be used to excite one or more samples in the sample wells.

E. Directional Emission Energy to Sensors Assay chip 2-110 contains one or more components per pixel and can improve the collection of emission energy by the sensor on the instrument. be. Such components may be designed to spatially direct the emissions energy towards the sensor and to increase the directionality of the emissions energy from sample wells 2-2111. Both surface optics and distant vision optics can be used to direct emission energy towards the sensor.

1. 1. The components within the assay chip 2-110 pixels, located near the sample wells of the surface optics pixels, can be configured to couple with the emission energy emitted by the sample. Such components can be formed at the interface between the two layers of the assay chip. For example, some emission energy coupling elements are formed at the interface between the sample well layer and the layer adjacent to the sample well layer on the opposite side of where the sample well is formed. obtain. In some cases, the layer below the sample well layer is a dielectric layer and the emission energy coupling element is capable of supporting the surface plasmon. In another embodiment, the sample well layer can be a conductive material adjacent to an optically transparent material. The surface energy coupling element can be a surface optical structure, which is excited by radiated emissions from the sample wells and with radiated emissions from the sample wells. Interact.

The characteristic dimensions of the surface optical structure, such as grating period, feature size, or distance from the sample well, put parallel components of the emission energy momentum vector into the surface wave momentum vector for the surface plasmon. May be selected for maximum coupling. For example, the parallel components of the emission energy momentum vector can be matched to the surface wave momentum vector of the surface plasmon supported by the structure according to some embodiments. In some embodiments, the distance d from the sample well to the edge or characteristic feature of the surface optical structure is, for example, perpendicular to the surface or angle θ from the direction perpendicular to the surface. It can be selected to orient the emission energy from the sample well in a selected direction, such as being tilted only. For example, the distance d can be an integer of the surface plasmon wavelength to direct the emissions perpendicular to the surface. In some embodiments, the distance d can be selected to be the surface plasmon wavelength of the fraction or the wavelength of its modulo.

According to some embodiments, the surface optical structure is capable of directing the radiated emission energy from the sample wells in a direction perpendicular to the sample well layer. The coupled energy can be oriented vertically in a narrowed directional radiation pattern.

An example of a surface optical structure is concentric grating. Concentric grating structures are formed within the pixels of the assay chip and are capable of directing emission energy towards one or more sensors in the pixel. Concentric grating structures can be formed around the sample wells. An example of a concentric circular grating surface 5-102 is shown in FIG. 5-1 as a surface plasmon structure. The circular grating can consist of any suitable number of rings, and the number of rings (6) shown in FIG. 10-1 is a non-limiting example. The circular grating can consist of a ring protruding from the surface of the conductive layer. For example, circular grating can be formed at the interface between the sample well layer and the dielectric layer formed beneath the sample well layer. The sample well layer can be a conductive material and concentric gratings can be formed by patterning the grating structure at the interface between the conductive material and the dielectric. Rings of circular grating can have regular periodic spacing, or can have irregular or aperiodic spacing between the rings. .. The sample well may be located at the center of the circular grating or near the center of the circular grating. In some embodiments, the sample wells can be positioned off the center of the circular grating and can also be positioned at a specific distance from the center of the grating. In some embodiments, the grating type emission coupling component can consist of spiral grating. An example of spiral grating 5-202 is shown in FIG. 5-2. The spiral grating 5-202 can consist of a spiral aperture in a conductive film. Any suitable dimension of spiral grating can be used to form the spiral grating.

FIG. 5-3 illustrates the radiation pattern 5-302 with respect to the emission energy from sample wells 2-211. The concentric grating structure 2-223 causes the emission energy to have a greater directionality compared to the radiation pattern formed in the absence of the grating structure 2-223. In some embodiments, the emission energy is directed downwards perpendicular to the metal layer 2-221.

Another example of a surface optics or surface plasmon structure is a nano-antenna structure. The nano-antenna structure can be designed to spatially direct the emission energy from the sample wells. In some embodiments, the location of the sample wells relative to the nanoantenna structure is selected to direct the emission energy from the sample wells in a particular direction towards one or more sensors. To. The nano-antenna can consist of a nano-scale dipole antenna structure so that the nano-scale dipole antenna structure produces a directional radiation pattern when excited by emission energy. It is designed. The nano-antenna can be dispersed around the sample well. A directional radiation pattern can result from the sum of the electromagnetic fields of the antenna. In some embodiments, a directional radiation pattern can result from the sum of the electromagnetic fields of the antenna, with the electromagnetic fields emitted directly from the sample. In some embodiments, the electromagnetic field emitted directly from the sample can be mediated by surface plasmons between the sample well and the nanoantenna structure.

The dimensions of the individual nano-antennas that form the nano-antenna structure can be selected so that the combined capabilities of the overall nano-antenna structure produce a particular distribution pattern. For example, the diameter of an individual nano-antenna can vary within the nano-antenna structure. However, in some cases the diameter can be the same within a set of nano-antennas. In other embodiments, several selected diameters can be used throughout the overall nano-antenna structure. Some nano-antennas can be dispersed over a circle of radius R and some can be radially shifted from the circle. Some nano-antennas can be evenly spaced around a circle of radius R (eg, centered on a uniform polar inclusion), and some are equal around a circle. Can be shifted from spacing. In some embodiments, the nano-antenna can be placed in a spiral configuration around the sample well. Additional or alternative, other configurations of nano-antennas are possible, such as matrix arrays around sample wells, cross-shaped distributions, and star-shaped distributions. Individual nano-antagons can have non-circular shapes such as squares, rectangles, crosses, triangles, bow ties, annular rings, pentagons, hexagons, polygons, and so on. In some embodiments, the perimeter of the aperture or disk can be an approximately multiple of an integer of fractional wavelength, for example (Ν / 2) λ.

The nano-antenna array can direct the emission energy from the sample into a concentrated radiation lobe. When the sample releases energy, it is possible to excite surface plasmons propagating from the sample wells to the nanoantennas dispersed around the sample wells. The surface plasmon can then excite a radiation mode or dipole emitter in the nanoantenna, which emits radiation perpendicular to the surface of the sample well layer. The mode or dipole phase excited in the nano-antenna will depend on the distance of the nano-antenna from the sample well. Choosing the distance between the sample wells and the individual nano-antennas controls the phase of the radiation emitted by the nano-antennas. The spatial radiation mode excited in the nano-antenna will depend on the geometry and / or size of the nano-antenna. Choosing the size and / or geometry of the individual nano-antennas controls the spatial radiation mode emitted by the nano-antennas. Contributions from all nano-antennas in the array, and in some cases sample wells, can determine one or more overall radiation lobes that form the radiation pattern. As can be understood, the phase and spatial radiation modes emitted from the individual nano-antennas can be wavelength dependent, with one or more global radiation lobes forming the radiation pattern. , It has become dependent on the wavelength. Numerical simulations of electromagnetic fields can be used to determine the overall radiation lobe pattern for emission energies of different characteristic wavelengths.

The nano-antenna can consist of an array of holes or apertures in a conductive membrane. For example, the nano-antenna structure can be formed at the interface between the conductive sample well layer and the underlying dielectric layer. The holes can consist of a set of holes dispersed within a concentric circle surrounding the center point. In some embodiments, the sample well is located at the center point of the array, but in other embodiments, the sample well may be off-center. Each set of circularly dispersed holes can consist of collections of different diameters arranged from minimum to maximum around a circular distribution. The hole diameter can vary between sets (eg, the smallest hole in one set can be larger than the smallest hole in another set), and the location of the smallest hole. Can be oriented at different polar angles for each set of circles. In some embodiments, one to seven sets of circularly dispersed holes can be present within the nanoantenna. In other embodiments, it is possible that there are more than seven sets. In some embodiments, the holes do not have to be circular and can be of any suitable shape. For example, the hole can be an ellipse, a triangle, a rectangle, and so on. In other embodiments, the distribution of holes does not have to be circular and can generate a spiral shape.

5-4A and 5-4B illustrate an exemplary nano-antenna structure consisting of holes or apertures in a conductive layer. FIG. 5-4A shows a top plan view of the surface of an assay chip comprising sample wells 5-108 surrounded by holes 5-122. The nano-antenna holes are distributed so that their centers are around a circle with a radius R approximately. In this non-limiting example, the hole diameter varies by increasing incrementally around the perimeter of the hole's circle. FIG. 5-4B shows a cross-sectional view of the assay chip shown in FIG. 5-4A along line BB. The sample well layer 5-116 can include sample wells 5-108 and apertures 5-122 that are part of the nanoantenna structure. The assay chip layer 5-118 extends beneath the sample well layer 5-116. Layers 5-118 can be a dielectric material and / or an optically transparent material.

In some embodiments, the nano-antenna structure can consist of multiple discs. The disk of the nano-antenna structure can be formed as a conductive disk protruding from the surface of the conductive material. The conductive material can be adjacent to an optically transparent material. In some embodiments, the nano-antenna can be dispersed around the sample well. In some cases, the nano-antennas may be distributed in a circle with radius R approximately around the sample wells. A nano-antenna array can consist of multiple sets of nano-antennas distributed around a sample well on a circle with approximately additional different radii.

5-5A and 5-5B illustrate an exemplary embodiment of a nano-antenna structure consisting of disks protruding from a conductive layer. FIG. 5-5A shows a schematic top plan view of the surface of an assay chip with sample wells 5-208 surrounded by a disk 5-224. The nano-antenna discs are dispersed around a circle with a radius of approximately R. In this non-limiting example, two diameters are used with respect to the disc, which alternates between these two diameters around the circumference of the nano-antenna circle. FIG. 5-5B shows a cross-sectional view of the assay chip shown in FIG. 6-3C along line CC'. The sample well layer 5-216 can include sample wells 5-208 and disc 5-224 that are part of the nanoantenna structure. The disc 5-224 protrudes from the sample well layer 5-216 by a specific distance. In some embodiments, the distance the disk extends from the sample well layer can vary within the nanoantenna structure. The assay chip layer 5-218 extends below the sample well layer 5-216. Layer 5-218 can be made of a dielectric material and / or an optically transparent material. The sample well layer 5-216 and the protruding disc can be a conductive material.

2. 2. Far-view optics In some embodiments, the layer immediately below the surface optics can be a spacer layer 2-225 of any suitable thickness, which is made from any suitable dielectric material. obtain. The spacer layer can be, for example, 10 μm thick and can be made from silicon dioxide. Alternatively, the spacer layer can be 48 μm or 50 μm. Underneath the spacer layer can be one or more lens layers with additional spacer layers. For example, FIG. 5-6A illustrates the upper lens layer 5-601, which can include at least one refracting lens. In some embodiments, the upper lens layer may be located 5 μm below the sample well layer 2-221. It is possible that there may be one or more lenses associated with each sample well. In some embodiments, a lens array may be used. In some embodiments, each lens of the upper lens layer 5-601 is centered below the sample well 2-211, and can have a radius less than, for example, 10.5 μm. be. The upper lens layer can be made from any suitable dielectric material, such as, but not limited to, silicon nitride.

The layer immediately below the upper lens layer can be a structural and / or optical layer 5-605 made from any suitable dielectric. This structural and / or optical layer 5-605 can be made from silicon dioxide in the form of fused silica. The layer immediately below the structural layer can be the lower lens layer 5-603 and the lower lens layer 5-603 can include at least one additional lens. In some embodiments, each lens in the lower lens layer 5-603 can also be centered below the sample well. The lower lens layer 5-603 can be made from any suitable dielectric material, such as, but not limited to, silicon nitride. The distance from the top of the upper lens layer to the bottom of the lower lens layer can be 100-500 μm. The layer immediately below the lower lens layer can include an antireflection layer, which allows both excitation energy and emission energy to pass and reduces the amount of reflected light. The layer immediately below the antireflection layer can contain structural components, allowing the chip to align with the device and mount on top of the device. The layer immediately below the chip mounting layer contains a protective cover and is capable of protecting the system from damage and contamination, including dust.

FIG. 5-6A illustrates the two lens layers using a refracting lens, but any suitable lens can be used. For example, Fresnel lenses, microlenses, refracting lens pairs, and / or flat lenses may be used. FIG. 5-6B uses Fresnel lenses in both the upper lens layer 5-611 and the lower lens layer 5-613 separated by structural and / or optical layers 5-605. The embodiment is illustrated.

In some embodiments, any of the interfaces between the layers described above in the chip can include an antireflection coating or an antireflection layer. Both the upper lens layer and the second lens layer are located below the sample wells and can focus the emission emitted from the array of sample wells into the relay lens of the instrument.

IV. Equipment components I. Microscope layer of the instrument In some embodiments, the instrument can include a microscope layer, which can include sublayers, as illustrated in FIG. 6-1. In particular, the microscope layer can include a sublayer containing a polychromic mirror 2-230 tilted by an angle θ to direct the excitation energy towards the assay chip. This polychromic mirror can be substantially dielectric, while substantially transmitting emission energy from the sample in one or more of the sample wells on the assay chip. Reflects the excitation energy. Optionally, an astigmatism correction element 6-101 containing an additional dielectric layer is provided beneath the polychromic mirror and at the same angle θ, but on an axis orthogonal to the axis of inclination of the polychromic mirror. Tilted around, it is possible to provide corrections for astigmatism caused by polychromic mirrors. In FIG. 6-1 the astigmatism correction element 6-101 is shown to be tilted in the same plane as the top filter, but the explanatory view shows the tilt with respect to the top filter. It should be recognized that it does not mean to limit the orientation of the astigmatism correction element 6-101. The astigmatism correction element 6-101 can also provide additional filtering. For example, the astigmatism correction element 6-101 can be another polychromic mirror that further filters the excitation energy while allowing the emission energy to pass through. A lens 6-103 can be provided under the astigmatism correction element 6-101 to further assist in processing the emission energy from the sample wells. Lens 6-103 can be, for example, 25.4 μm in diameter, but any suitable diameter can be used. In some embodiments, the lens is a relay lens consisting of a plurality of lens elements. For example, a relay lens can include six separate lens elements. In some embodiments, the relay lens can be approximately 17.5 mm in length. Additional filtering elements can be used before or after lens 6-103 to further reject the excitation energy and prevent it from reaching the sensor.

A. Emission energy emitted from the sample in the sensor chip sample well can be transmitted to the pixel's sensor in various ways, some examples of which are described in detail below. Some embodiments use optical and / or plasmonic components to direct light of a particular wavelength to a region or portion of a sensor specialized to detect light of a particular wavelength. It is possible to increase the possibility of The sensor can include multiple sub-sensors for simultaneously detecting emission energies of different wavelengths.

FIG. 6-2A is a schematic diagram of a single pixel of a sensor chip according to some embodiments, in which at least one sorting element 6-127 is used to provide emission energy of a particular wavelength. Direct to each sub-sensor 6-111 to 6-114. Emission energy 2-253 travels from the sample well through the assay chip and the optical system of the instrument until it reaches the sorting element 6-127 of the sensor chip. Sorting element 6-127 couples the wavelengths of emission energy 2-253 to spatial degrees of freedom, thereby separating the emission energy into its constituent wavelength components, which is the sorted emission. It is called energy. In Figure 6-2A, the emission energy 2-253 is split through the dielectric material 6-129 into four sorted emission energy paths, each of which is a pixel sub-sensor. It is schematically illustrated that it is associated with 6-111 to 6-114. In this way, each sub-sensor is associated with a different part of the spectrum, forming a spectrometer for each pixel of the sensor chip.

Any suitable sorting element 6-127 can be used to separate different wavelengths of emission energy. Embodiments can use optical elements or plasmonic elements. Examples of optical sorting elements include, but are not limited to, holographic gratings, phase mask gratings, amplitude mask gratings, and offset Fresnel lenses. Examples of plasmonic sorting elements include, but are not limited to, phased nano-antenna arrays and plasmonic quasicrystals.

FIG. 6-2B is a schematic diagram of a single pixel of the sensor chip according to some embodiments, in which filtering elements 6-121 to 6-124 are used and the emission energy of a particular wavelength. To each sub-sensor to prevent emission energy of other wavelengths from reaching the other sub-sensor. Emission energy 2-253 travels from the sample well through the assay chip and the optical system of the instrument until it reaches one of the filtering elements 6-121 to 6-124. Filtering elements 6-121 to 6-124 may be associated with specific segments of sub-sensors 6-11 to 6-114, respectively, and filtering elements 6-121 to 6-124 may provide emission energy. By absorbing (not shown in FIG. 6-1B) and / or by reflecting the emission energy, the emission energy of each wavelength is transmitted, and the emission of other wavelengths. -Each is configured to reject energy. After passing through each filtering element, the filtered emission energy travels through the dielectric material 6-129 and collides with the corresponding sub-sensors 6-111 to 6-114 of the pixel. Thus, each sub-sensor is associated with a different portion of the spectrum, forming a spectrometer for each pixel of the sensor chip.

Any suitable filtering element can be used to separate different wavelengths of emission energy. Embodiments can use optical filtering elements or plasmonic filtering elements. Examples of optical sorting elements include, but are not limited to, reflective multilayer dielectric filters or absorbent filters. Examples of plasmonic sorting elements include, but are not limited to, selectivity surfaces designed to transmit energy at specific wavelengths, and photonic band-gap crystals.

Alternatively, or in addition to the sorting and filtering elements described above, additional filtering elements may be installed adjacent to the respective sub-sensors 6-11 to 6-114. Additional filtering elements can include a thin loss film, which is configured to generate intensifying interference with respect to the emission energy of a particular wavelength. The thin lossy membrane can be a single membrane or a multilayer membrane. The thin lossy membrane can be made from any suitable material. For example, a thin lossy film can be made from a material having a refractive index n approximately the same as the extinction coefficient k. In another embodiment, the thin loss film can be made from a material whose index of refraction n is within about two orders of magnitude from the value of the material's extinction coefficient k. Non-limiting examples of such materials at visible wavelengths are germanium and silicon.

The thin lossy membrane can be of any suitable thickness. In some embodiments, the thin lossy membrane can be 1-45 nm thick. In other embodiments, the thin lossy membrane can be 15-45 nm thick. In yet another embodiment, the thin lossy membrane can be 1-20 nm thick. In FIG. 6-3A, the thin lossy films 6-211 to 6-214 each have a different thickness at least partially determined by the wavelength associated with each sub-sensor 6-11 to 6-114. The embodiment is illustrated. The thickness of the membrane determines, at least in part, the distinct wavelength that will selectively pass through the thin lossy membrane and reach the sub-sensor. As illustrated in FIG. 6-221, the thin lossy film 6-221 has a thickness d1 and the thin lossy film 6-212 has a thickness d2, resulting in a thin loss. The sex film 6-213 has a thickness d3 and the thin lossy film 6-214 has a thickness d4. The thickness of each subsequent thin lossy film is smaller than that of the previous thin lossy film, so that d1> d2> d3> d4.

Additional or alternative, thin lossy membranes can be formed from different materials with different qualities so that emission energies of different wavelengths intensify and interfere with each other in each sub-sensor. For example, the index of refraction n and / or the extinction coefficient k may be selected to optimize the transmission of emission energy at a particular wavelength. FIG. 6-3B illustrates thin lossy films 6-221 to 6-224 with the same thickness, but each thin lossy film is made of a different material. In some embodiments, both the thin lossy membrane material and the thin lossy membrane thickness can be selected so that the emission energies of the desired wavelengths intensify and interfere with each other and are transmitted through the membrane.

FIG. 6-1 illustrates an embodiment in which a combination of diffractive elements and lenses is used to sort emission energy by wavelength. The first layer 6-105 of the sensor chip can include blazed phase gratings. Blazed gratings can be, for example, blaze at an angle φ that is substantially equal to 40 degrees, and the line spacing of the blazed grating (Λ) can be substantially equal to 1.25 μm. .. Those skilled in the art will appreciate that different blaze angles and periodicities can be used to achieve the separation of light of different wavelengths of emission energy. Moreover, any suitable diffractive optics can be used to separate wavelengths with different emission energies. For example, phase masks, amplitude masks, blazed gratings, or offset Fresnel lenses can be used.

The second layer 6-106 of the sensor chip 2-260 may include one or more Fresnel lenses, and the one or more Fresnel lenses may include one or more Fresnel lenses of the first layer 6-105. Arranged below, it further sorts the emission energy and directs the emission energy to the sensor 6-107. Moreover, any suitable lens element can be used to further separate wavelengths with different emission energies. For example, a refracting lens can be used in place of a Fresnel lens.

The various components of FIG. 6-1 can be spaced apart at any suitable distance. For example, the surface of the sensor may be located at a distance of 5 μm under the Fresnel lens layer 6-106. The distance from the center of the lens 6-103 of the microscope layer to the Fresnel lens layer 6-106 can be 50.6 mm. The blazed phase grating 6-105 can be located at a distance of approximately 100 μm above the surface of the sensor. Alternatively, the distance from the bottom of the assay chip to the top of the grating 6-105 can be approximately 53 mm. The width of the sensor layer can be approximately 10 mm.

The various layers of the assay chip and instrument do not have to be in the order described above. In some embodiments, the focusing element and / or the sorting element, as well as the imaging optics of the instrument, can be in reverse order. For example, the blazed phase grating 6-105 may be installed after the Fresnel lens layer 6-106. Alternatively, the focusing element and / or the sorting element and the imaging optics can be integrated into a single diffractive optical element (DOE). In addition, various components of the assay chip and instrument can be mixed, for example, imaging optics can occur both above and below the focusing element and / or the sorting element.

Any of the interfaces between the layers described above in the system, including the interface between the air and the layers of the system, can include an antireflection coating.
B. Embodiments of Optical Blocks of Equipment In some embodiments, the optical blocks of equipment 1-120 can include some or all of the optical components described above. be. Optical blocks can provide optical components such as those located in Figure 6-4. In addition to the components described above, the optical block can include a first fiber connector 6-401 and a second fiber connector 6-402, a first fiber connector. A first optical fiber carrying a first wavelength of excitation energy can be connected to 6-401, and a second wavelength of excitation energy is carried to a second fiber connector 6-402. A second optical fiber can be connected. By way of example, and without limitation, the first excitation wavelength of the excitation energy can be 630-640 nm. The fiber optic connector can be any suitable conventional connector, such as an FC connector or an LC connector. If two different wavelengths are input, the wavelengths can be combined by a wavelength combiner 6-403, such as a dichroic mirror or a polychromic mirror. The second excitation wavelength can be 515-535 nm. The input excitation energy can be any suitable polarization, such as linear polarization. In some embodiments, the fiber carrying the excitation energy can be a polarization retaining fiber. Optionally, an excitation filter and a polarizing element such as a fiber optic to free space coupler can be used after the fiber optic input to further filter or modify the properties of the excitation energy.

The optical block includes one or more metal housings and is capable of holding lenses and other optical components for optical processing such as beam shaping. FIG. 6-4 illustrates four metal housings 6-405 to 6-408, each holding a lens and / or other optical component. It is possible that there can be any number of lenses used to collimate and focus the excitation energy. One or more mirrors 6-411 and 6-421 are located between some of the metal housings to guide the excitation energy towards assay chip 2-110. In FIG. 6-4, the first mirror 6-411 directs the excitation energy from the second housing 6-406 to the third housing 6-407, and the second mirror 6-412 is the second. Excitation energy is reflected from the housing 6-408 of No. 4 to the polychromic dielectric mirror 2-230. The polychromic dielectric mirror 2-230 directs the excitation energy towards the astigmatism correction filter 6-601.

In some embodiments, circular polarization can be directed into the sample well, causing the luminescent marker to emit luminescence with similar intensities. A quarter wavelength plate can be used to transfer linearly polarized light to circularly polarized light before it reaches the assay chip. The polychromic dielectric mirror 2-230 directs the excitation energy to the quarter wavelength plate 6-415. As illustrated in FIG. 6-4, the quarter wavelength plate 6-415 may be disposed between the astigmatism correction filter 6-101 and the assay chip 2-110. The circularly polarized excitation energy is then directed towards multiple pixels on the assay chip. Excitation energies that are not directed towards the pixel can be absorbed by the beam dump component 6-417. The excitation energy that reaches the sample inside one or more sample wells will cause the sample to emit emission energy, which is directed towards sensor 2-260. Emission energy can pass through optical components such as polarization optics, astigmatism correction elements 6-101, polychromic mirrors 2-230, and relay lenses 6-103. The polychromic mirror acts as a filter, which can be, for example, a notch filter, a spike filter, or a cut-off filter. The relay lens 6-103 can image the emission energy toward the sensor. A portion of the emission energy can then pass through one or more emission filters 6-421 and 6-422 located above the sensor 2-260 and one or more. Emission filters 6-421 and 6-422 are capable of further filtering emission energy. In some embodiments, the emissions filter can be tilted at an angle with respect to the incident emission energy propagation direction to tune the transmission characteristics of the filter and / or reduce the interference caused by back reflections. It is designed to let you. If the top filter 6-421 is tilted at an angle θ, the bottom filter 6-422 is tilted at the same angle θ but around an axis orthogonal to the axis of tilt of the top filter and emits. It is possible to ensure that astigmatism does not occur in the radiation beam path.

C. Sensors The present disclosure provides various embodiments of sensors, sensor operations, and signal processing methods. According to some embodiments, the sensor 2-122 in the pixels of the sensor chip 2-260 can and receives emission energy from one or more tags in the sample well. It can consist of any suitable sensor capable of producing one or more electrical signals representing the emitted emissions energy. In some embodiments, the sensor can consist of at least one photodetector (eg, a pn junction formed within a semiconductor substrate). 7-1A and 7-1B show one embodiment of a sensor that can be made within pixel 2-100 of the sensor chip.

According to some embodiments, the sensor 2-122 can be formed at each pixel 2-100 of the sensor chip. The sensor may be associated with sample wells 2-211 of the assay chip. It is possible that one or more transparent layers 7-110 may be present above the sensor, allowing emissions from the sample wells to travel to the sensor with little attenuation. Sensors 2-122 can be formed in the semiconductor substrate 7-120 at the pixel base, according to some embodiments, and are positioned on the same side of the sample well as the assay chip (not shown). Can be done.

The sensor can consist of one or more semiconductor junction photodetector segments. Each semiconductor junction can consist of a first conductive type well. For example, each semiconductor junction can consist of n-type wells formed within a p-type substrate, as shown in the drawings. According to some embodiments, the sensor 2-122 can be arranged as a bullseye detector 7-162, as shown in the plan view of FIG. 7-1B. The first photodetector 7-124 may be located in the center of the sensor and the second annular photodetector 7-122 may surround the central photodetector. Electrical contact to the wells can be made through the conductive traces 7-134 formed at the first or subsequent level of metallization and through the conductive vias 7-132. It is possible that there is a region of the highly doped semiconductor material 7-126 in the contact region of the via. In some embodiments, the field oxide 7-115 can be formed on the surface between the photodetectors and can cover a portion of each photodetector. In some embodiments, it is possible that there are additional semiconductor devices 7-125 (eg, transistors, amplifiers, etc.) formed within the pixel adjacent to the sensor 2-122. It is possible that there are additional metallization levels 7-138, 7-136 within the pixel.

In some embodiments, the metallization level 7-136 can extend across most of the pixels and also has a centered opening above the photodetector 7-124. It is possible to have and allow emissions from the sample well to reach the sensor. In some cases, metallization levels 7-136 can serve as a reference potential or ground plane, and additionally serve as an optical block, at least some background radiation. It is possible to prevent (eg, radiation from an excitation source or ambient environment) from reaching sensor 2-260.

As shown in FIGS. 7-1A and 7-1B, sensors 2-122 go to multiple photodetector segments 7-122, 7-124 that are spatially and electrically separated from each other. And can be further divided. In some embodiments, the segment of sensor 2-122 can consist of a region of conversely doped semiconductor material. For example, a first charge storage well 7-124 for the first sensor segment is formed by doping a first region of the substrate and into the first well a first conductive type (eg, n). -It is possible to have a type). The substrate can be p-type. A second charge storage well 7-122 for the second sensor segment can be formed by doping a second region of the substrate and have a first conductive type within the second well. be. The first and second wells can be separated by the p-type region of the substrate.

Multiple segments of sensors 2-122 may be arranged in any suitable manner other than the bullseye layout, and it is possible that there are three or more segments within the sensor. For example, in some embodiments, multiple photodetector segments 7-142 are laterally separated from each other to form a striped sensor 7-164 as shown in FIG. 7-1C. Is possible. In some embodiments, the quad (or quadrant) sensor 7-166 may be formed by arranging the segments 7-144 in a quad pattern, as shown in FIG. 7-1D. In some embodiments, the arc segment 7-146 is formed in combination with the bullseye pattern and segmented into an arc, as shown in FIG. 7-1E. It is possible to form 168. Another sensor configuration can consist of a pie piece section, which can include individual sensors arranged in separate circular sections. In some cases, the sensor segments may be placed symmetrically around the sample wells 2-211, or asymmetrically around the sample wells. The placement of the sensor segments is not limited to the above-mentioned placement, and any suitable distribution of the sensor segments may be used.

The inventor says that the quadrant sensor 7-166, pie sector sensor, or similar sector sensor can more preferably be reduced to a smaller pixel size than other sensor configurations. Was found. Quadrant and sector detectors can consume less pixel area and active sensor area for multiple wavelengths detected. Sensors can be arranged in a variety of geometric configurations. In some examples, the sensors are arranged in a square or hexagonal configuration.

The sensors of the present disclosure may be addressable independently (or individually). Individually addressable sensors can detect the signal and provide an output independent of other sensors. Individually addressable sensors can be individually readable.

In some embodiments, the stacked sensors 7-169 are made by making a plurality of isolated sensor segments 7-148 into a vertical stack, as shown in FIG. 7-1F. Can be formed. For example, the segments may be positioned one above the other, with or without an insulating layer between the stacked segments. Each vertical layer can be configured to absorb emissions of a particular energy and to pass emissions at different energies. For example, the first detector is capable of absorbing and detecting shorter wavelength radiation (eg, radiation with a blue wavelength of about 500 nm or less from a sample). The first detector is capable of passing green and red wavelength emissions from the sample. The second detector is capable of absorbing and detecting green wavelength radiation (eg, between about 500 nm and about 600 nm) and allowing it to pass red emissions. The third detector is capable of absorbing and detecting red emissions. In some embodiments, a reflective film 7-149 is incorporated into the stack and is capable of reflecting light in the selected wavelength band and returning it through the segment. For example, the membrane can reflect radiation of green wavelength that was not absorbed by the second segment and can be returned through the second segment to increase its detection efficiency.

In some embodiments with vertically stacked sensor segments, the emissions coupling component is not included in the sample wells and is a separate spatial distribution pattern of sample emissions that depends on the emission wavelength. It is possible to create. Discrimination of spectrally different emissions is achieved using vertically-stacked sensors 7-169 by analyzing the proportion of signals from the stacked segments, according to some embodiments. obtain.

In some embodiments, the segment of sensor 2-122 is formed from silicon, but any suitable semiconductor (eg, Ge, GaAs, SiGe, InP, etc.) may also be used. In some embodiments, the sensor segment can consist of an organic photoconducting film. In other embodiments, quantum dot photodetectors may be used for the sensor segment. Quantum dot photodetectors can respond to different emission energies based on the size of the quantum dots. In some embodiments, multiple quantum dots of various sizes can be used to discriminate between different emission energies or wavelengths received from the sample wells. For example, the first segment may be formed from quantum dots having a first size, and the second segment may be formed from quantum dots having a second size. In various embodiments, sensors 2-122 can be formed using conventional CMOS processes.

As described above, the emission coupling component may be made adjacent to the sample well in some embodiments. Sorting elements 2-243 can modify emissions from the samples in sample wells 2-211, creating a separate spatial distribution pattern of sample emissions that depends on the emission wavelength. FIG. 7-2A shows an example of the first spatial distribution pattern 7-250, which can be produced from the first sample at the first wavelength. The first spatial distribution pattern 7-250 may have a prominent central lobe oriented towards the central segment of the bullseye sensor 7-162, for example as shown in FIG. 7-2B. It is possible. Such a pattern 7-250 can be produced from any suitable diffractive element, where the sample emits at a wavelength of about 663 nm. The projected pattern 7-252 incident on the sensor can appear as illustrated in FIG. 7-2B.

FIG. 7-2C shows a spatial distribution pattern 7-260, which, according to some embodiments, can be produced from a second sample emitting at a second wavelength from the same sample well. .. The second spatial distribution pattern 7-260 consists of two lobes of radiation and can be different from the first spatial distribution pattern 7-250. The projected pattern 7-262 of the second spatial distribution pattern 7-260 can appear as shown in FIG. 7-2D, according to some embodiments. A second spatial distribution pattern 7-260 can be produced from any suitable diffractive element, where the sample emits at a wavelength of about 687 nm.

The segments of sensors 2-122 may be arranged to detect specific emission energies, according to some embodiments. For example, emissions coupling structures and sensor segments adjacent to sample wells may be designed in combination to increase signal differentiation between specific emission energies. Emission energy can correspond to the selected tag that will be used with the sensor chip. As an example, the bullseye sensor 7-162 is capable of sizing and / or positioning its segment to better match the patterns 7-260, 7-262 projected from the sample. The higher intensity area is more centered within the active segment of the sensor. Alternatively or additionally, the diffractive element may be designed to alter the projected patterns 7-260, 7-262 so that the strong region fits more centrally within the sensor segment. ing.

Sensor 2-122 can consist of two segments, but in some embodiments it is possible to discriminate between three or more spectrally distinct emission bands from the sample. For example, each emission band can create a separate projected pattern on top of the sensor segment, creating a separate combination of signals from the sensor segment. The combination of signals can be analyzed to discriminate and identify emission bands. 7-2E to 7-2H represent the results from a numerical simulation of the signal from the two-segment sensors 2-122 exposed in four separate emission patterns. As can be seen, each combination of signals from the two sensor segments is separate and can be used to discriminate between emitters at four wavelengths. For the simulation, the detector segment outside the bullseye sensor 7-162 had a larger area, so more signals were accumulated for that detector. In addition, light colliding in the area between the detectors can generate carriers, which can drift towards one of the detector segments and contribute to the signal from both segments. ..

In some embodiments, it is possible that there are N photodetector segments per pixel, where N can be any integer value. In some embodiments, N can be greater than or equal to 1 and less than or equal to 10. In other embodiments, N can be 2 or more and 5 or less. The number M of discriminable sample emissions (eg, separate emission wavelengths from different luminescent tags) that can be detected by N detectors can be N or greater. The determination of M sample emissions can be achieved by assessing the proportion of signals from each sensor segment, according to some embodiments. In some embodiments, the ratio, sum, and / or amplitude of the received signal can be measured and analyzed to determine the characteristic wavelength of emissions from the sample wells.

In some embodiments, two or more emitters are capable of emitting different characteristic wavelengths within a given time window within sample wells 2-211. Sensors 2-122 can simultaneously detect signals from multiple emissions at different wavelengths and provide a summed signal for data processing. In some embodiments, multi-wavelength emissions may be distinguishable as another set of signal values from the sensor segment (eg, different from those shown in FIGS. 7-2E to 7-2H). Signal value). The signal value can be analyzed to determine that multi-wavelength emissions have occurred and to identify a particular combination of emitters associated with the emissions.

The present inventor also considered and analyzed a bullseye sensor having four concentric segments. Signals from the segments are plotted in FIGS. 7-2I and 7-2J with respect to the same emission conditions associated with FIGS. 7-2G and 7-2H, respectively. Also, the 4-segment bullseye sensor shows an identifiable signal that can be analyzed to identify a particular emitter in the sample well.

When wavelength filtering is used in each sensor segment, or when spectral separation is high, each segment of the sensor is capable of substantially detecting only the selected emission band. For example, the first wavelength can be detected by the first segment, the second wavelength can be detected by the second segment, and the third wavelength can be detected by the third segment.

With reference to FIG. 7-1A again, it is possible that there is an additional electronic circuit 7-125 within pixel 2-100, which collects and reads signals from each segment of sensor 2-122. Can be used for. 7-3A and 7-3D show circuits that can be used in combination with multi-segment sensors, according to some embodiments. As an example, the signal acquisition circuit 7-310 can consist of three transistors for each sensor segment. The arrangement of the three transistors is shown in FIG. 7-3B, according to some embodiments. At charge storage nodes 7-311 associated with each segment, the signal level can be reset by the reset transistor RST, and the signal level for the segment (determined by the amount of charge at the charge storage node) is read. It can be read by the transistor RD.

Pixel circuits can further include amplified and correlated double sampling circuits 7-320, according to some embodiments. Amplification and double sampling circuits are configured with transistors that are configured to amplify the signal from the sensor segment and, for example, when no emission energy is present on the sensor (eg, the excitation energy in the sample well). With a transistor configured to reset the voltage level at the charge storage node (before application) and to read the background or "reset" signal at the node, and to read the subsequent emission signal. It is possible to consist of.

According to some embodiments, correlated double sampling is used to reduce background noise by subtracting the background or reset signal level from the detected emission signal level. The collected emission and background signals associated with each segment of the sensor can be read over parallel lines 7-330. In some embodiments, the emission signal level and background signal are time-division-multiplexed over a common column line. It is possible that there will be separate columns for each sensor segment. The signal from the parallel line can be buffered and / or amplified by amplification circuit 7-340, which can be located outside the active pixel array, and provided for further processing and analysis. In some embodiments, the subtraction of the double sampled signal is calculated off-chip, for example by a system processor. In other embodiments, the subtraction can be done on the chip or in the circuit of the base device.

Some embodiments of correlated double sampling can be operated by selecting a row for the sample, and the sensor associated with the row has the signal charge accumulated over the sampling period and the signal level. contains. The signal level can be read simultaneously on the parallel line. After sampling the integrated signal level, all pixels in the selected row are reset and can be sampled immediately. This reset level can be correlated with the next signal to be accumulated, which means that the accumulation will start after the reset is released and the frame time will be accumulated later when the same row is selected again. finish. In some embodiments, the frame reset value can be stored off-chip so that the stored correlated reset value can be deducted when the signal finishes accumulating and is sampled.

In some embodiments, the sensor 2-122 with three or more segments may require additional circuitry. FIG. 7-3C shows a signal acquisition 7-312, amplification 7-320, and double sampling circuit associated with a quad sensor. According to some embodiments, signals from more than one segment can be time-division-multiplexed in pixels over a common signal channel, as shown in the drawings. The time-division-multiplexed signal can include background signals sampled for each segment for noise elimination. Additionally, signals from more than one segment can be time-division-multiplexed over a common column line.

According to some embodiments, temporal signal acquisition techniques are used to reduce background signal levels from one or more excitation sources and / or different emissions from different emitters associated with the sample. Can be used to determine. FIG. 7-4A shows, according to some embodiments, fluorescence emissions and attenuation from two different emitters that can be used to tag the sample. The two emissions have visibly different time decay characteristics. The first time decay curve 7-410 from the first emitter can correspond to a common fluorescent molecule such as rhodamine. The second time decay curve 7-420 can be a characteristic of the second emitter, such as a quantum dot or phosphorescent emitter. Both emitters show an emission decay tail that extends for some time after the emitter's initial excitation. In some embodiments, the signal acquisition technique applied during the emission attenuation tail is, in some embodiments, to reduce the background signal from the excitation source, and in some embodiments, Timing can be chosen to distinguish between emitters.

According to some embodiments, time-delayed sampling is used during the emission attenuation tail to reduce the background signal due to radiation from the excitation source. 7-4B and 7-4C illustrate time-delayed sampling according to some embodiments. FIG. 7-4B shows the temporal evolution of the excitation pulse 7-440 of the excitation energy from the excitation source and the subsequent emission pulse 7-450 that can be obtained from the sample excited in the sample well. Shows. The excitation pulse 7-440 can be generated as a result of driving the excitation source by the drive signal 7-442 over a short period of time, as shown in FIG. 7-4C. For example, the drive signal can start at the _first time t1 and end at the _second time t2. The duration of the drive signal (t ₂ -t ₁ ) can be between about 1 picosecond and about 50 nanoseconds, according to some embodiments, but in some embodiments. Shorter durations can also be used.

_At time t3 following the end of the drive signal for the excitation source, the sensor _3-260 (or sensor segment) at the pixel is during a _second time interval extending from time t3 to time t4. In addition, it may be controlled by a gate to store charge at the charge storage node 7-311. The second time interval can be between about 1 nanosecond and about 50 microseconds, according to some embodiments, but in some embodiments other durations are also used. obtain. As can be seen with reference to FIG. 7-4B, the charge storage node will collect more signal charge due to the ejecting sample and then due to the excitation source. Therefore, an improved signal-to-noise ratio can be obtained.

Referring again to FIG. 7-4A, the corresponding signal in the sensor can peak at different times due to the different temporal emissions characteristics of the emitter. In some embodiments, the signal-acquisition technique applied during the emission attenuation tail can be used to discriminate between different emitters. In some embodiments, temporal detection techniques are used in combination with spatial and spectral techniques (eg, as described above in connection with Figure 7-2) with different emitters. It is possible to distinguish.

Figures 7-4D through 7-4H show how double sampling in a sensor or sensor segment can be used to distinguish between two emitters with different temporal emissions characteristics. It is shown in the figure. FIG. 7-4D shows the emission curves 7-470 and 7-475 associated with the first emitter and the second emitter, respectively. As an example, the first emitter can be a common fluorophore, such as rhodamine, and the second emitter can be a quantum dot or phosphorescent emitter.

FIG. 7-4E represents a dynamic voltage level at charge storage nodes 7-311 that can occur in response to the two different emission characteristics of FIG. 7-4D. In the example, the first voltage curve 7-472 corresponding to the fluorescent emitter changes more rapidly due to the shorter emission span, and at the _first time t1, its maximum (or node polarity). Depending on, it is possible to reach the minimum). The second voltage curve 7-477 may change more slowly due to the longer emissions characteristics of the second emitter and reach its maximum (or minimum) at the _second time t2. It is possible.

In some embodiments, sampling of charge storage nodes can be done at _two times t3 _, t4 after sample excitation, as shown in FIG. 7-4F. For example, the first read signal 7-481 may be applied to read the first voltage value from the charge storage node at the _first time t3. After that, the second read signal 7-482 does not reset the charge storage node between the first read and the second read, and at the _second time t4, the second read from the charge storage node. Can be applied to read out voltage values. Analysis of the two sampled signal values is then used to determine which of the two emitters provided the detected signal level.

FIG. 7-4G is an example of two signals from a first read and a second read that can be obtained for a first emitter having an emission curve 7-470 as shown in FIG. 7-4D. Shows. FIG. 7-4H is an example of two signals from a first read and a second read that can be obtained for a second emitter having an emission curve 7-475 as shown in FIG. 7-4D. Shows. For example, the sampling sequence shown in FIG. 7-4F for the first emitter will sample curves 7-472 and obtain approximately the same value at the two read times. In the case of the second emitter, the sampling sequence shown in FIG. 7-4F samples two different values of curve 7-477 at two read times. The resulting pair of signals from the two read times distinguishes between the two emitters and can be analyzed to identify each emitter. Also, according to some embodiments, double sampling for background subtraction may be performed to subtract the background signal from the first and second read signals.

In operation, the sensor 2-260 on the sensor chip can undergo a wavelength calibration procedure prior to data acquisition from the sample to be analyzed. The wavelength calibration procedure may include causing the sensor to receive different known energies with characteristic wavelengths that may or may not correspond to the fluorofore wavelengths that may or may not correspond to the fluorofore wavelengths that may be used with the sensor chip. It is possible. Different energies can be applied in the sequence and the calibration signal can be recorded from the sensor for each energy. The calibration signal can then be stored as a reference signal, which determines to process the actual data acquisition and which one or more emission wavelengths are detected by the sensor. Can be used for.

V. Luminescent Marker Embodiments can use any suitable luminescent marker to label a sample (eg, a single molecule) within the sample being analyzed. In some embodiments, commercially available fluorophores may be used. By example, and not by limitation, the following fluorophores, Atto Rho 14 (“ATRho14”), Dylight 650 (“D650”), SetaTau 647 (“ST647”), CF 633 (“C633”), CF 647: ("C647"), Alexa fluoro 647 ("AF647"), BODIPY 630/650 ("B630"), CF 640R ("C640R"), and / or Atto 647N ("AT647N") can be used. Additional and / or optionally, the luminescent marker can be modified in any suitable manner to increase the speed and accuracy of the sample analysis process. For example, a light stabilizer can be conjugated to a luminescent marker. Examples of light stabilizers include, but are not limited to, oxygen scavengers or triplet state quenchers. Conjugating a light stabilizer to a luminescent marker can increase the rate of emitted photons and also reduce the "blinking" effect when the luminescent marker does not emit photons. It is possible. In some embodiments, increasing the rate of photon emissions can increase the likelihood of detecting biological events when biological events occur on the millisecond scale. Increasing the rate of photon events can then increase the signal-to-noise ratio of the emission signal and increase the rate at which the measurement is being made, leading to faster and more accurate sample analysis.

VI. Excitation Source Excitation Source 2-250 can be any suitable source that is arranged to deliver excitation energy to at least one sample well 2-111 of the assay chip. The pixel above the assay chip can be a passive source pixel. The term "passive source pixel" is used to describe a pixel in which excitation energy is delivered from the outer region of the pixel or assay chip pixel array to the pixel, for example, the excitation can be in the instrument. It is used.

According to some embodiments, the excitation source is capable of exciting the sample via a radiation process. For example, the excitation source has visible radiation (eg, radiation with a wavelength between about 350 nm and about 750 nm), near-infrared radiation (eg, wavelength between about 0.75 micron and about 1.4 micron). Radiation) and / or short wavelength infrared radiation (eg, radiation having a wavelength between about 1.4 microns and about 3 microns) in at least one excitation region of at least one sample well of the assay chip. It is possible to provide in 3-215. In some embodiments, the radiation excitation source is a material consisting of a medium (eg, a molecule, a quantum dot, or a selected molecule and / or a quantum dot) that is directly adjacent to the excitation region of the sample well. It is possible to provide energy to excite the layer). The mediator is capable of transferring its energy to the sample via a non-radiative process (eg, via FRET or DET).

In some embodiments, the excitation source can provide two or more sources of excitation energy. For example, a radiated excitation source can deliver excitation energies with two or more distinct spectral characteristics. As an example, a multicolor LED can emit energy focused on more than one wavelength, and these energies can be delivered to the excited region of the sample well.

In overview, and according to some embodiments, the instrument comprises at least one excitation source 2-250 and excites or excites into at least one excitation region of at least one sample well of the assay chip. It is possible to provide the excitation energy to at least one mediator that converts the energy or couples the excitation energy to at least one sample in one or more excitation regions. As shown in FIG. 2-3, the radiation excitation energy 2-251 from the excitation source 2-250 can collide with, for example, the region around the sample well 2-211. In some embodiments, it is possible to have an excitation coupling structure 2-223 that assists in concentrating the incident excitation energy within the excitation region 2-215 of the sample well.

The excitation source can be characterized by one or more distinct spectral bands, each with a characteristic wavelength. An example of spectral emissions from an excitation source is shown in the spectral graph of FIG. 8-1A for educational purposes only. The excitation energy may be substantially contained within the spectral excitation bands 8-110. The peak wavelength 8-120 of the spectral excitation band can be used to characterize the excitation energy. The excitation energy can also be characterized by a spectral distribution, eg, a full width at half maximum (FWHM) value as shown in the drawings. Excitation sources that produce energy as shown in FIG. 8-1A can be characterized as delivering energy at a wavelength of approximately 540 nm radiation and as having a FWHM bandwidth of approximately 55 nm.

FIG. 4-1B shows the spectral characteristics of one excitation source (or multiple excitation sources) capable of providing two excitation energy bands to one or more sample wells. According to some embodiments, as illustrated in the drawings, the first excitation band 8-112 is at approximately 532 nm and the second excitation band 8-114 is at approximately 638 nm. In some embodiments, the first excitation band can be at approximately 638 nm and the second excitation band can be at approximately 650 nm. In some embodiments, the first excitation band can be at approximately 680 nm and the second excitation band can be at approximately 690 nm. According to some embodiments, the peak of the excitation band can be within ± 5 nm of these values.

In some cases, the radiated excitation source is capable of producing a wide excitation band, as shown in FIG. 8-1A. The broad excitation band 8-110 can have a bandwidth greater than approximately 20 nm, according to some embodiments. A wide excitation band can be created, for example, by light emitting diodes (LEDs). In some embodiments, the radiated excitation source is capable of creating a narrow excitation band, as shown in FIG. 8-1B. The narrow excitation band can be created, for example, by a laser diode or by spectrally filtering the output from the LED.

In some embodiments, the excitation source can be a light source. Any suitable light source can be used. Some embodiments are capable of using a source of incoherent light, while others are capable of using a source of coherent light. By way of example, and without limitation, incoherent light sources according to some embodiments are such as organic LEDs (OLEDs), quantum dots (QLEDs), nanowire LEDs, and (inorganic) organic semiconductor LEDs. , It is possible to include different types of light emitting diodes (LEDs). By example, and without limitation, coherent light sources according to some embodiments are organic lasers, quantum dot lasers, vertical cavity surface emitting lasers (VCSELs), end face emitting lasers, and distributed feedback lasers. It is possible to include different types of lasers, such as DFB) laser diodes. Additional or alternative, slab-coupled optical waveguide lasers (SCOWLs), or other asymmetric single-mode waveguide structures can be used. Additional or alternative, solid state lasers such as Nd: YAG or Nd: glass excited by a laser diode or flashlamp may be used. Additional or alternative, laser diodes excited by fiber lasers may be used. In some embodiments, the output of the laser excitation source is half the wavelength in a nonlinear crystal, or periodic polarization inversion lithium niobate (PPRN), or other similar periodic polarization inversion nonlinear crystal. On the other hand, the frequency can be doubled. This frequency doubling process can allow the efficient use of lasers to generate wavelengths that are more suitable for excitation. There can be more than one type of excitation source for an array of pixels. In some embodiments, different types of excitation sources can be combined. Excitation sources can be made according to conventional techniques used to make selected types of excitation sources.

The characteristic wavelength of the source of the excitation energy can be selected based on the selection of the luminescent marker used in the assay analysis. In some embodiments, the characteristic wavelength of the source of the excitation energy is selected for the direct excitation of the selected fluorophore (eg, single photon excitation). In some embodiments, the characteristic wavelength of the source of the excitation energy is selected for indirect excitation (eg, polyphoton excitation, or harmonic conversion to a wavelength that will provide direct excitation). In some embodiments, the excitation energy can be generated by a light source configured to generate the excitation energy at a particular wavelength for application to sample wells. In some embodiments, the characteristic wavelength of the excitation source can be smaller than the characteristic wavelength of the corresponding emissions from the sample. In some embodiments, the characteristic wavelength of the excitation source can be greater than the characteristic wavelength of emissions from the sample, and the excitation of the sample can occur through polyphoton absorption.

The excitation source can include a battery or any other power source, which can be located somewhere other than the integrated bioanalytical device. For example, the excitation source can be positioned within the instrument and the power can be coupled to the integrated bioanalytical device via conductive wires and connectors.

VIII. Usage, device operation, and user interface Device 2-120 may be controlled using software and / or hardware. For example, the device may be controlled using processing devices 1-123, such as ASICs, FPGAs and / or general purpose processors running software.

FIG. 9-1 illustrates a flow chart of the operation of the device 2-120 according to some embodiments. After the user has obtained the sample for analysis, the user initiates a new analysis in Acts 9-101. This may be done by providing instructions to device 2-120 via user interface 2-125, for example by pressing a button. In Acts 9-103, instrument 2-120 checks whether assay chip 2-110 from a previously performed analysis is still inserted into instrument 2-120. If it is determined that an old chip is present, power to the excitation source may be turned off in Acts 9-105 and the user in Acts 9-107 of User Interface 2-125. Prompted to remove the previous chip using the indicator, device 2-120 waits for the chip to be removed in actions 9-109.

When the previous chip is removed by the user, or if device 2-120 determines in actions 9-103 that the previous chip has already been removed, the user in actions 9-111. You will be prompted to insert a new assay chip 2-110 for a new analysis. Instrument 2-120 then waits for a new assay chip 2-110 to be inserted in Act 9-113. When the user inserts a new chip, in Act 9-115, the user interface 2 so that the sample to be analyzed is placed on the exposed top surface of assay chip 2-110. Prompted by the -125 indicator and also prompted to close the lid over device 2-120. Instrument 2-120 then waits for the lid to be closed in Act 9-117. When the lid is closed by the user, in Act 9-119, the excitation source is driven to produce excitation energy to excite the sample portion of the sample present in the sample well of assay chip 2-110. Can be done. In Acts 8-121, the emission energy from the sample is detected by sensor 2-122 and the data from sensor 2-122 is passed to processing device 2-123 for analysis. In some embodiments, the data may be streamed to an external computing device 2-130. In act 2-123, device 2-120 checks whether the data collection is complete. Data acquisition could be completed after a certain length of time, identifying a particular number of excitation pulses from the excitation source, or one particular target. When the data collection is complete, the data analysis ends at 9-125.

FIG. 9-2 illustrates an exemplary self-calibration routine with some embodiments. The calibration routine can be performed at any suitable time prior to sample analysis. For example, it can be done once by the manufacturer for each device prior to shipping to the end user. Alternatively, the end user can perform the calibration at any suitable time. As discussed above, equipment 2-120 is capable of distinguishing between emission energies with different wavelengths emitted from different samples. Instrument 2-120 and / or computing device 2-130 are associated with, for example, each particular color of light associated with a luminescent tag used to tag the molecule of the sample being analyzed. Can be calibrated by calibration. In this way, the exact output signal associated with a particular color can be determined.

To calibrate the device, calibration samples associated with a single luminescent tag are provided to instrument 2-120 one by one. When the user places a sample consisting of a luminescent tag that emits emission energy of a single wavelength on assay chip 2-110 and inserts assay chip 2-110 into instrument 2-120. , Self-calibration begins at Acts 9-201. Using user interface 2-125, the user instructs instrument 2-120 to initiate self-calibration. In response, in Act 8-203, instrument 2-120 measures single wavelength emission energy from the calibration sample and by irradiating assay chip 2-110 with excitation energy. Run the calibration analysis. Instrument 2-120 is then capable of saving the detection pattern measured on the sub-sensor array of sensors 2-122 for each pixel of the sensor array in Acts 9-205. The detection pattern for each luminescent tag can be thought of as the detection signature associated with the luminescent tag. Thus, the signature can be used as a training data set used to analyze data received from unknown samples analyzed in subsequent analysis runs.

The above calibration routine can then be performed for all calibration samples associated with a single luminescent tag. Thus, each sensor 2-122 in the array of pixels is associated with calibration data, which is then performed in actions 9-207 after the completion of the calibration routine. Can be used to determine the luminescent tag present in the sample well during the analysis of.

FIG. 9-3 further illustrates how calibration data can be acquired and used to analyze the data, according to some embodiments. In Acts 9-301, calibration data is obtained from the sensor. This can be done using the self-calibration routine described above. In Acts 9-303, a transformation matrix is generated based on the calibration data. The transformation matrix maps sensor data to the emission wavelengths of the sample and is also an mxn matrix, where m is the number of luminescent tags with different emission wavelengths and n is the emission per pixel. The number of sub-sensors used to detect energy. Therefore, each column of the transformation matrix represents the calibration value for the sensor. For example, if there are 4 sub-sensors per pixel and 5 different luminescent tags, the transformation matrix is a 4x5 matrix (ie, 4 rows and 5 columns), where each column is , Associated with different luminescent tags, the values in the column correspond to the measurements obtained from the sub-sensors during the self-calibration routine. In some embodiments, each pixel can have its own transformation matrix. In other embodiments, calibration data from at least some of the pixels can be averaged, and then all pixels can use the same transformation matrix based on the averaged data. ..

In Acts 9-305, analytical data associated with the bioassay is obtained from the sensor. This can be done by any of the methods described above. In Acts 9-307, the wavelength of the emission energy and / or the identity of the luminescent tag can be determined using the transformation matrix and analytical data. This can be done in any suitable manner. In some embodiments, the analytical data is multiplied by the pseudoinverse of the transformation matrix, resulting in an mx1 vector. The luminescent tag associated with the vector component having the maximum value can then be identified as the luminescent tag present in the sample well. Embodiments are not limited to this technique. In some embodiments, there are restrictions, such as least squares methods or maximum likelihood techniques, to prevent possible pathologies that can occur when the inverse of a matrix with small values is taken. An optimization routine may be performed to determine the luminescent tag present in the sample well.

The aforementioned method of using calibration data to analyze data from the sensor can be performed by any suitable processor. For example, processing device 2-123 of device 2-120 can perform the analysis, or computing device 2-130 can perform the analysis.

IX. Computing Devices FIG. 10 illustrates an example of a suitable computing system environment 1000 on which embodiments may be implemented. For example, the computing device 2-130 of FIG. 2-1 may be implemented according to the computing system environment 1000. Additionally, the computing system environment 1000 can serve as a control system, which is programmed to control the instrument to perform the assay. For example, the control system can control the excitation source to emit light and direct the light towards the sample well of the assay chip, and one of the sample wells. The sensor can be controlled to allow detection of emission light from one or more samples, and the signal from the sensor, for example, by analyzing the spatial distribution of emission energy. Can be analyzed to identify the sample present in the sample well. The computing system environment 1000 is merely an example of a suitable computing environment and is not intended to propose any limitation on the scope of use or functionality of the present invention. No computing environment 1000 should be construed as having any dependency or requirement for any one or combination of components illustrated in the exemplary operating environment 1000.

The embodiments can work with a number of other general purpose or dedicated computing system environments or configurations. Examples of well-known computing systems, environments, and / or configurations that may be suitable for use with the present invention are, but are not limited to, personal computers, server computers, handheld or laptop computers. Devices, multiprocessor systems, microprocessor-based systems, set-top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, and any of the above systems or devices. Including distributed computing environment and so on.

The computing environment is capable of executing computer-executable instructions such as program modules. In general, a program module includes routines, programs, objects, components, data structures, etc. that perform a particular task or perform a particular extracted data type. The invention can also be practiced in a distributed computing environment, where tasks are performed by remote processing devices linked through communication networks. In a distributed computing environment, program modules can be located in both local and remote computer storage media, including memory storage devices.

Referring to FIG. 10, an exemplary system for carrying out the present invention includes a general purpose computing device in the form of a computer 1010. The components of computer 1010 can include, but are not limited to, a processing unit 1020, system memory 1030, and system bus 1021, which can include various system components including system memory. Coupling to processing unit 1020. The system bus 1021 is one of several types of bus structures, including memory buses or memory controllers, peripheral buses, and local buses that use any of the various bus architectures. Is possible. As an example, and not exclusively, such architectures include Industry Standard Architecture (ISA) Bus, Micro Channel Architecture (MCA) Bus, Extended ISA (EISA) Bus, Video Electronics Standards. Includes Association (VESA) Local Bus and Peripheral Component Interconnect (PCI) Bus, also known as Mezanin Bus.

The computer 1010 typically includes various computer readable media. A computer-readable medium is any available medium that can be accessed by the computer 1010, including both volatile and non-volatile media, removable and non-removable media. By way of example, and without limitation, the computer readable medium can consist of a computer storage medium and a communication medium. Computer storage media are volatile and non-volatile media, removable, implemented by any method or technique with respect to the storage of information, such as computer readable instructions, data structures, program modules, or other data. Includes both non-removable and non-removable media. Computer storage media are, but are not limited to, RAM, ROM, EEPROM, flash memory, or other memory technologies, CD-ROMs, digital versatile disks (DVDs), or other optical disk storage, magnetic cassettes. , Magnetic tape, magnetic disk storage, or other magnetic storage device, or any other medium that can be used to store desired information and can be accessed by computer 1010. The communication medium typically embodies other data in a modulated data signal, such as computer-readable instructions, data structures, program modules, or, for example, carrier waves or other transport mechanisms, and any information. Includes delivery medium. The term "modulated data signal" means a signal having one or more of its properties set or altered in a manner that encodes the information in the signal. By way of example, and without limitation, the communication medium is a wired medium, such as a wired network or a direct wired connection, as well as an acoustic medium, an RF medium, an infrared medium, and other wireless media, and the like. Includes wireless media. Also, any combination of the above should be included within the scope of computer readable media.

The system memory 1030 includes computer storage media in the form of volatile and / or non-volatile memory, such as read-only memory (ROM) 1031 and random access memory (RAM) 1032. Is possible. The basic input / output system 1033 (BIOS) contains basic routines that help transmit information between elements in the computer 1010, such as during boot, and the basic input / output system 1033 (BIOS). ) Is typically stored in ROM 1031. The RAM 1032 typically contains a data and / or program module, the data and / or the program module having immediate access to the processing unit 1020 and / or currently being operated by the processing unit 1020. There is. By way of example, and not by limitation, FIG. 10 illustrates an operating system 1034, an application program 1035, other program modules 1036, and program data 1037.

Computer 1010 can also include other removable / non-removable volatile / non-volatile computer storage media. As a mere example, FIG. 10 shows a hard disk drive 1041 that reads and writes to a non-removable non-volatile magnetic medium, a magnetic disk drive 1051 that reads and writes to a removable non-volatile magnetic disk 1052, and a CD ROM. Alternatively, an optical disk drive 1055 that reads and writes to a removable non-volatile optical disk 1056, such as another optical medium, is illustrated. Other removable / non-removable volatile / non-volatile computer storage media that can be used in an exemplary operating environment are, but are not limited to, magnetic tape cassettes, flash memory cards, and digital versatility. Includes disks, digital videotapes, solid-state RAM, and solid-state ROM. The hard disk drive 1041 is typically connected to system bus 1021 through a non-removable memory interface such as interface 1040, and the magnetic disk drive 1051 and optical disk drive 1055 are typically. In particular, it is connected to the system bus 1021 by a removable memory interface such as interface 1050.

The drives and their associated computer storage media, discussed above and illustrated in FIG. 10, provide computer-readable instructions, data structures, program modules, and storage of other data about the computer 1010. do. In FIG. 10, for example, the hard disk drive 1041 is illustrated as storing an operating system 1044, an application program 1045, another program module 1046, and program data 1047. These components can be either the same as or different from the operating system 1034, application program 1035, other program modules 1036, and program data 1037. Please note that. The operating system 1044, the application program 1045, the other program modules 1046, and the program data 1047 are given different numbers here, illustrating that they are, at a minimum, different copies. .. Users can enter commands and information into computer 1010 through input devices such as the keyboard 1062 and a pointing device 1061 commonly referred to as a mouse, trackball, or touchpad. Is. Other input devices (not shown) can include microphones, joysticks, game pads, satellite dishes, scanners, and the like. These input devices and other input devices may be connected to the processing unit 1020 through the user input interface 1060 coupled to the system bus, which may be a parallel port, a game port, or a universal serial port. Often connected by other interfaces and bus structures, such as buses (USB). Also, a monitor 1091 or other type of display device is connected to the system bus 1021 via an interface such as the video interface 1090. Also, in addition to the monitor, the computer can include other peripheral output devices such as speakers 1097 and printer 1096, which can be connected through the output peripheral interface 1095.

Computer 1010 can be operated in a networked environment using a logical connection to one or more remote computers, such as remote computer 1080. The remote computer 1080 can be a personal computer, server, router, network PC, peer device, or other common network node, and only the memory storage device 1081 is shown in FIG. Although illustrated in, a remote computer 1080 typically includes many or all of the elements described above with respect to the computer 1010. The logical connection shown in FIG. 10 includes a local area network (LAN) 1071 and a wide area network (WAN) 1073, but may include other networks as well. Such networking environments are common in offices, enterprise-wide computer networks, intranets, and the Internet.

When used in a LAN networking environment, the computer 1010 is connected to the LAN 1071 through a network interface or adapter 1070. When used in a WAN networking environment, the computer 1010 typically includes a modem 1072, or other means for establishing communication through the WAN 1073, such as the Internet. The modem 1072 can be internal or external, and the modem 1072 can be connected to system bus 1021 via user input interface 1060, or other suitable mechanism. In a networked environment, the program module shown for computer 1010 or a portion thereof may be stored in a remote memory storage device. As an example and, but not as a limitation, FIG. 10 illustrates the remote application program 1085 as being on top of the memory device 1081. It will be appreciated that the network connections shown are exemplary and other means of establishing communication links between computers may also be used.

VIII. Conclusion Although some embodiments of at least one embodiment of the present invention have been described, it is understood that various alternatives, modifications, and improvements will be readily conceivable to those of skill in the art. Should be.

Such alternatives, modifications, and improvements are intended to be part of this disclosure and are intended to be within the spirit and scope of the invention. Further, although the advantages of the present invention have been shown, it should be recognized that not all embodiments of the present invention will include all of the described advantages. Some embodiments may not implement any of the features described herein and in some cases as advantageous. Therefore, the above description and drawings are merely examples.

The above-described embodiment of the present invention can be carried out by any of a number of methods. For example, embodiments may be implemented using hardware, software, or a combination thereof. When implemented in software, the software code is any suitable processor, or processor, whether it is located in a single computer or distributed among multiple computers. Can be performed on the collection of. Such processors can be implemented as integrated circuits and include one or more processors in an integrated circuit component, such as a CPU chip, GPU chip, microprocessor, microprocessor, or coprocessor. Includes commercially available integrated circuit components known in the art by name. Alternatively, the processor may be implemented in a custom circuit, such as an ASIC, or a semi-custom circuit that results from configuring a programmable logic device. As a further alternative, the processor, whether commercially available, semi-custom, or custom, can be part of a larger circuit or semiconductor device. As a particular example, some commercially available microprocessors have a plurality of cores, one or a subset of which can constitute the processor. However, the processor can be implemented using any suitablely formatted circuit.

Furthermore, it should be recognized that computers can be embodied in any of a number of forms, such as rack-mounted computers, desktop computers, laptop computers, or tablet computers. In addition, computers generally have the appropriate processing capabilities, including personal digital assistants (PDAs), smart phones, or any other suitable portable or fixed electronic device. Can be embedded in devices that are not considered.

Also, the computer can have one or more input and output devices. These devices can be used, among other things, to present a user interface. Examples of output devices that can be used to provide a user interface include a printer or display screen for the visual representation of the output, and a speaker or other sound generating device for the audible representation of the output. Examples of input devices that can be used with respect to user interfaces include keyboards as well as pointing devices such as mice, touchpads, and digitizing tablets. As another example, the computer can receive input information through voice recognition or in other audible formats.

Such computers may be interconnected in any suitable manner by one or more networks, including local area networks or wide area networks, such as corporate networks or the Internet. Such networks can be based on any suitable technology, can operate according to any suitable protocol, and can include wireless networks, wired networks, or fiber optic networks. It is possible.

Also, the various methods or processes outlined herein are encoded as software that can be run on one or more processors using any one of the various operating systems or platforms. obtain. Additionally, such software can be written using either a number of suitable programming languages and / or programming tools or scripting tools, and can also be run on a framework or virtual machine. Can be compiled as executable machine language code or intermediate code.

In this regard, the invention is one or the other that implements the methods of implementing the various embodiments of the invention discussed above when run on one or more computers or other processors. Computer-readable storage media (or computer-readable media) encoded by multiple programs (eg, computer memory, one or more floppy disks, compact disks (CDs), optical disks, digital discs. It can be embodied as a video disk (DVD), magnetic tape, flash memory, circuit configuration within a field programmable gate array or other semiconductor device, or other tangible computer storage medium). As is clear from the previous example, a computer-readable storage medium can retain information for a sufficient amount of time to provide a non-temporary form of computer-executable instructions. Such one or more computer-readable storage media may be transportable, on which one or more programs may be loaded onto one or more different computers or other processors. It has become possible to implement various aspects of the invention as discussed above. As used herein, the term "computer-readable storage medium" includes only computer-readable media that may be considered a product (ie, manufactured product) or machine. Alternatively or additionally, the invention may be embodied as a computer-readable medium other than a computer-readable storage medium, such as a propagating signal.

The term "program" or "software" can be used to program a computer or other processor to implement various aspects of the invention as discussed above, computer code or computer. As used herein, in a general sense, to represent any set of executable instructions. Additionally, according to one embodiment of this embodiment, one or more computer programs that implement the methods of the invention, when executed, need to be on a single computer or processor. Instead, it should be recognized that in order to implement various aspects of the invention, it can be distributed in a modular fashion among a plurality of different computers or processors.

Computer-executable instructions can be in many forms, such as program modules, executed by one or more computers or other devices. In general, a program module includes routines, programs, objects, components, data structures, etc. that perform a particular task or perform a particular extracted data type. Typically, the functionality of the program module can be combined or distributed as needed in various embodiments.

Also, the data structure can be stored in any suitable form on a computer readable medium. For simplicity of illustration, a data structure may be shown to have relevant fields through locations within the data structure. Such relationships can be similarly achieved by allocating storage for the fields to a location in a computer-readable medium that conveys the relationships between the fields. However, any suitable mechanism can be used to establish relationships between information within fields of a data structure, other than establishing relationships between pointers, tags, or data elements. Including through the use of the mechanism of.

Various aspects of the invention may be used alone, in combination, or in various arrangements not specifically discussed in the embodiments described above, and thus in their application. It is not limited to the details and arrangement of the components described in the above description or illustrated in the drawings. For example, the embodiments described in one embodiment may be combined with the embodiments described in the other embodiments in any manner.

Further, the present invention can be embodied as a method, and an example thereof is provided. The actions performed as part of the method can be ordered in any suitable manner. Thus, embodiments may be constructed in which the actions are performed in a different order than shown, which may be shown as continuous actions in the exemplary embodiment, but may perform several actions simultaneously. It is possible to include doing.

The use of prejudicial terms such as "first", "second", "third", etc. to modify the claims element within the claims is different in itself. It does not imply any priority, order, or order of one claim element with respect to any claim element, nor does it imply a temporal order in which the act of the method is performed. Simply, one claim element with a particular name is used as a label to distinguish it from another element with the same name (without the use of predicate terms) to distinguish the claim elements.

Also, the linguistic expressions and terminology used herein are for explanatory purposes only and should not be considered limiting. "Including,""comprising," or "having,""contining,""involving," and variations thereof are used herein. By doing so means to include the items listed thereafter and their equivalents, as well as additional items.

Claims (18)

An array of sample wells provided on the surface of the chip, each sample well in said array being formed to accept one sample.
A plurality of lenses formed in the chip, each of the plurality of lenses is formed so as to collect light emitted from one sample well in the array, and the plurality of lenses are formed. A device comprising a plurality of lenses, each of which is a refracting lens having a convex surface close to the array of sample wells and a plane far from the array of said sample wells. The device of claim 1, wherein each of the plurality of lenses corresponds to an individual sample well in the array. The device of claim 2, wherein each of the plurality of lenses is arranged to direct light emitted from a corresponding sample well in the array towards the sensor. The device of claim 2, wherein each of the plurality of lenses overlaps a corresponding sample well in the array. The device of claim 4, wherein each of the plurality of lenses is centered below the corresponding sample well. The device of claim 5, wherein each of the plurality of lenses is provided 5 μm below the corresponding sample well. The device according to claim 1, wherein the plurality of lenses are provided on the lens layer of the chip. 7. The apparatus of claim 7, further comprising a layer of dielectric material provided between the lens layer and the array of sample wells. The apparatus according to claim 8, wherein the layer of the dielectric material contains silicon oxide, and the lens layer contains silicon nitride. The apparatus according to claim 8, wherein the layer of the dielectric material has a thickness of 5 μm. An array of sample wells provided on the surface of the chip, each sample well in said array being formed to accept one sample.
A plurality of lenses formed in the lens layer of the chip, wherein each lens of the plurality of lenses is formed so as to collect light emitted from one sample well in the array. Lens and
A layer of dielectric material provided between the lens layer and the array of sample wells, wherein the dielectric material is made of silicon oxide and the lens layer is made of a layer of dielectric material containing silicon nitride. Become a device. A step of forming an array of a plurality of sample wells provided on the surface of a chip, wherein each sample well in the array is formed to receive one sample. The process of forming and
A step of forming a plurality of lenses in the chip, wherein the plurality of lenses are formed so as to collect light emitted from one sample well in the array, and among the plurality of lenses. Manufacture of an apparatus comprising the steps of forming a plurality of lenses, each of which is a refracting lens having a convex surface close to the array of sample wells and a plane far from the array of sample wells. Method. The step of forming the plurality of lenses includes a step of arranging each lens of the plurality of lenses so as to direct the light emitted from the corresponding sample wells in the array toward the sensor. , The manufacturing method according to claim 12. The step of forming the plurality of lenses includes a step of forming each lens so that each lens of the plurality of lenses overlaps with a corresponding sample well in the array. 13. The manufacturing method according to 13. In the step of forming the array of the plurality of sample wells, the step of forming the individual sample wells so that the individual sample wells in the array are aligned with the corresponding lenses of the plurality of lenses. 14. The manufacturing method according to claim 14. 12. The manufacturing method according to claim 12, further comprising a step of forming a layer of a dielectric material provided between the plurality of lenses and an array of sample wells. The step of forming the plurality of sample wells is
A step of forming a laminate of a material consisting of at least one metal layer above an optically transparent layer,
12. The production method according to claim 12, further comprising a step of etching at least through a laminate of the materials to form a plurality of sample wells. 17. The manufacturing method according to claim 17, wherein the etching step further comprises a step of etching a side wall portion in which each sample well of the plurality of sample wells is tapered.

Images